AUTOREFERAT Dr Przemyslaw Szafranski‚. 4 Autoreferat wersja ang. P.Sz_..pdfAUTOREFERAT Dr Przemyslaw Szafranski Department of Molecular and Human Genetics, Baylor College of Medicine,

P. Szafranski Załącznik 4

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AUTOREFERAT

Dr Przemyslaw Szafranski

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas,

USA

Houston, 2019

TABLE OF CONTENTS

1. Name…….……………………………………………………………………….................…..2

2. Diplomas and scientific degrees.………………………………………………….….....….….2

3. Employment information……………………………………………..……………………….2

4. Indication of achievement resulting from art. 16 sec. 2 of the Act of 14 March 2003 on

academic degrees and academic title, and on degrees and title in the field of art ……….…….3

4.1 Title of scientific achievement ………………………………………………….............….3

4.2 List of publications constituting scientific achievements referred to in art. 16 sec. 2 acts….3

4.3 Grants awarded for research, within which the work included in the scientific achievement

was created ……………………………………………………………………………….…….5

4.4 Discussion of the scientific purpose of the abovementioned work and its results together

with discussion of their possible use ……………………………………………………….…...5

5. Other academic achievements of the habilitant……...………………...………………..….15

5.1 List of publications in magazines in the Journal Citation Reports database obtained after the

doctoral dissertation, which are not part of the performance mentioned in point 4……………15

5.2 List of other publications written after the doctorate……………….…………….………..24

5.3 List of patents……………………………………………………………………..……….24

5.4 Discussion of other scientific and research achievements ………………………………..24

5.5 Bibliometric indicators ………………………………………………………………...….26

5.6 Awards and distinctions for scientific activity……………………………………….…....26

6. Didactics and popularization of science………………………….…………………….…....26


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1. NAME

Przemyslaw Szafranski

2. DIPLOMAS AND SCIENTIFIC DEGREES

1984 Doctor of natural sciences in the field of biochemistry

Instytute of Biochemistry and Biophysics Polish Akademy of Sciences in Warsaw

The title of the dissertation: The model of substrate binding in the catalytic center

of Escherichia coli RNA polymerase during the initiation of transcription

Promoter: prof. dr hab. Kazimierz L. Wierzchowski

1978 Master in Biology in Molecular Biology

Faculty of Biology, University of Warsaw in Warsaw

Promoter: prof. dr hab. Zbigniew Kaniuga

3. EMPLOYMENT INFORMATION

od 2013 Assistant Professor

Department of Molecular and Human Genetics, Baylor College of

Medicine, Houston, Texas, USA

2009-2013 Staff Scientist

Department of Molecular and Human Genetics, Baylor College of Medicine,

Houston, Texas, USA

1997-2009 Postdoctoral Research Associate

Departments of Pediatrics (Section of Cardiology), and Pathology, Baylor

College of Medicine, Houston, Texas, USA

1993-1997 Postdoctoral Research Associate

Department of Biomedical Engineering, Boston University, Boston,

Massachusetts, USA

1986-1993 Adiunkt

Department of Biophysics, Institute of Biochemistry and Biophysics PAS in

Warsaw

1984-1986 Postdoctoral Fellow

Department of Biochemistry, New York University Medical Center, New

York City, New York, USA


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4. INDICATION OF ACHIEVEMENT resulting from art. 16 sec. 2 of the Act of 14 March

2003 on academic degrees and academic title, and on degrees and title in the field of art

(Journal of Laws of 2017, item 1789)

4.1 Title of scientific achievement

Non-coding aspects of the molecular genetics of the dysplasia ACDMPV

4.2 The list of publications constituting the scientific achievement referred to in art. 16 sec.

2 acts

(1) Schulze KV*, Szafranski P*, Lesmana H, Hopkin RJ, Hamvas A, Wambach JA, Shinawi M,

Zapata G, Carvalho CMB, Liu Q, Karolak JA, Lupski JR, Hanchard NA, Stankiewicz P (2019)

Novel parent-of-origin-specific differentially methylated loci on chromosome 16. Clinical

Epigenetics, 11(1):60. doi: 10.1186/s13148-019-0655-8.

*Equal contribution

IF2017=6.091, MNiSW=30, number of citations=0

(2) Szafranski P, Kośmider E, Liu Q, Karolak JA, Currie L, Parkash S, Kahler SG, Roeder E,

Littlejohn RO, DeNapoli TS, Shardonofsky FR, Henderson C, Powers G, Poisson V, Bérubé D,

Oligny L, Michaud JL, Janssens S, De Coen K, Van Dorpe J, Dheedene A, Harting MT, Weaver

MD, Khan AM, Tatevian N, Wambach J, Gibbs KA, Popek E, Gambin A, Stankiewicz P (2018)

LINE- and Alu-containing genomic instability hotspot at 16q24.1 associated with recurrent and

nonrecurrent CNV deletions causative for ACDMPV. Human Mutation, 39(12):1916-1925. doi:

10.1002/humu.23608. PMID: 30084155

IF2017=5.359, MNiSW=40, number of citations =0

(3) Szafranski P*, Karolak JA*, Lanza D*, Gajęcka M, Heaney J, Stankiewicz P (2017)

CRISPR/Cas9-mediated deletion of lncRNA Gm26878 in the distant Foxf1 enhancer region.

Mammalian Genome, 28(7-8):275-282. doi: 10.1007/s00335-017-9686-7. PMID: 28405742

IF2017=2.687, MNiSW=25, liczba cytowań=4

* Equal contribution

(4) Szafranski P, Herrera C, Proe LA, Coffman B, Kearney DL, Popek E, Stankiewicz P (2016)

Narrowing the FOXF1 distant enhancer region on 16q24.1 critical for ACDMPV. Clinical

Epigenetics, 8:112. PMID: 27822317


(5) Szafranski P, Gambin T, Dharmadhikari AV, Akdemir KC, Jhangiani SN, Schuette J,

Godiwala N, Yatsenko SA, Sebastian J, Madan-Khetarpal S, Surti U, Abellar RG, Bateman DA,

Wilson AL, Markham MH, Slamon J, Santos-Simarro F, Palomares M, Nevado J, Lapunzina P,

Chung BH, Wong WL, Chu YW, Mok GT, Kerem E, Reiter J, Ambalavanan N, Anderson SA,


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Kelly DR, Shieh J, Rosenthal TC, Scheible K, Steiner L, Iqbal MA, McKinnon ML, Hamilton SJ,

Schlade-Bartusiak K, English D, Hendson G, Roeder ER, DeNapoli TS, Littlejohn RO, Wolff DJ,

Wagner CL, Yeung A, Francis D, Fiorino EK, Edelman M, Fox J, Hayes DA, Janssens S, De Baere

E, Menten B, Loccufier A, Vanwalleghem L, Moerman P, Sznajer Y, Lay AS, Kussmann JL,

Chawla J, Payton DJ, Phillips GE, Brosens E, Tibboel D, de Klein A, Maystadt I, Fisher R, Sebire

N, Male A, Chopra M, Pinner J, Malcolm G, Peters G, Arbuckle S, Lees M, Mead Z, Quarrell O,

Sayers R, Owens M, Shaw-Smith C, Lioy J, McKay E, de Leeuw N, Feenstra I, Spruijt L, Elmslie

F, Thiruchelvam T, Bacino CA, Langston C, Lupski JR, Sen P, Popek E, Stankiewicz P (2016)

Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins. Human

Genetics, 135(5):569-586. doi: 10.1007/s00439-016-1655-9. PMID: 27071622


(6) Szafranski P, Dharmadhikari AV, Wambach JA, Towe CT, White FV, Grady RM, Eghtesady

P, Cole FS, Deutsch G, Sen P, Stankiewicz P (2014) Two deletions overlapping a distant FOXF1

enhancer unravel the role of lncRNA LINC01081 in etiology of alveolar capillary dysplasia with

misalignment of pulmonary veins. American Journal of Medical Genetics A, 164A(8):2013-2019.

doi: 10.1002/ajmg.a.36606. PMID: 24842713


(7) Szafranski P, Yang Y, Nelson MU, Bizzarro MJ, Morotti RA, Langston C, Stankiewicz P

(2013) Novel FOXF1 deep intronic deletion causes lethal lung developmental disorder, alveolar

capillary dysplasia with misalignment of pulmonary veins. Human Mutation, 34(11):1467-1471.

doi: 10.1002/humu.22395. PMID: 23943206


(8) Szafranski P, Dharmadhikari AV, Brosens E, Gurha P, Kolodziejska KE, Zhishuo O, Dittwald

P, Majewski T, Mohan KN, Chen B, Person RE, Tibboel D, de Klein A, Pinner J, Chopra M,

Malcolm G, Peters G, Arbuckle S, Guiang SF 3rd, Hustead VA, Jessurun J, Hirsch R, Witte DP,

Maystadt I, Sebire N, Fisher R, Langston C, Sen P, Stankiewicz P (2013) Small noncoding

differentially methylated copy-number variants, including lncRNA genes, cause a lethal lung

developmental disorder. Genome Research, 23(1):23-33. doi: 10.1101/gr.141887.112. PMID:

23034409


Impact factor (IF) was given based on the Journal Citation Reports (JCR) database, the number of

citations was based on the Web of Science (WoB) database. Total IF of the above eight

publications amounts to 44,894, the total number of their MNiSW points is 270, the total number

of their previous citations is 127. Copies of the above publications can be found in attachment no.

6. Statements of the habilitant and co-authors regarding their contribution to the creation of each

of the abovementioned publications can be found in attachment no. 7.


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4.3 Grants awarded for research, within which the work included in the scientific

achievement was created

(1) National Organization for Rare Disorders (NORD), Modelling ACDMPV therapies by

targeting negative regulators of FOXF1 and genes outside the SHH pathway, Grant Number: 2016

NORD Grant 16001, 09.14.2017-09.14.2019, Rola w projekcie: kierownik (ang. principal

investigator, PI)

(2) National Institutes of Health (NIH), Epigenomic dysfunction at 16q24.1: vascular defects and

perinatal consequences, Grant Number: R01HL137203, 2017-2022, Rola w projekcie:

wykonawca (ang. co-investigator)

(3) National Organization for Rare Disorders (NORD), Towards designing ACDMPV therapy:

Deciphering epigenetic regulation of FOXF1, Grant Number: 2014 NORD Grant, 03.11.2015-

03.10.2017, Rola w projekcie: kierownik

(4) National Organization for Rare Disorders (NORD), Long non-coding RNAs as potential

diagnostic and therapeutic targets in patients with Alveolar Capillary Dysplasia with

Misalignment of Pulmonary Veins (ACD/MPV), Grant Number: 2012 NORD grant, 12.21.2012-

12.21.2014, Rola w projekcie: kierownik

(5) National Institutes of Health (NIH), Pathogenetics of the FOX transcription factor gene cluster

on 16q24.1, Grant Number: R01HL101975-03, 05.01.2010-04.30.2014, Rola w projekcie:

wykonawca

4.4 Discussion of the scientific purpose of the abovementioned work and its results together

with discussion of their possible use

4.4.1 Introduction and purpose of the conducted research

Only about 2% of the human genome directly encodes proteins, but a significant fraction of its

remaining non-coding part also undergoes at least residual transcription to unconventional RNAs,

for many of which there is evidence of their specific biological functions. The importance of non-

coding DNA in the expression of genes and etiology of genetic diseases is further emphasized by

the fact that nucleotide variants segregating with an abnormal phenotype very often map to non-

coding sequences. Still another unexpected result of sequencing of the human genome was the

realization that almost half of it is composed of retroelements, mainly LINE1, SINE Alu and

HERV. In addition, the group of evolutionarily young retrotransposons, L1HS, retained the ability

to actively move in the genome. Retrotransposons are the internal mutagen of the genome being

able to modify the structure and expression of genes, and, like many other repetitive sequences,

are substrates in non-allelic homologous recombination (NAHR) or inaccurate repair of DNA

breaks leading to deletions, duplication (CNV), inversion and translocation.


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The aim of the research presented here, published in the eight original papers included in the

scientific achievement (point 4.2), was to identify non-coding sequences of the human genome,

important from the point of view of the etiology of congenital lung developmental disorder,

alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV, MIM # 265380)

and to evaluate the possibilities of eventual future therapeutic use of the obtained results.

ACDMPV is a rare, almost always fatal disease of newborns (the incidence is around 1 in 100,000

live births), for which the only alternative is a lung transplant (Slot et al 2018). Its main symptoms

include respiratory failure and pulmonary hypertension. From the histopathological point of view,

it is characterized primarily by underdevelopment of the blood-air barrier and the pulmonary

vascular system in general (Fig. 1).

Fig. 1. The image of abnormally developed lung tissue in ACDMPV. Hypertrophy of the pulmonary arteriolar

muscular layer (a), thickened septa between the pulmonary alveoli (*), reduced number of capillaries (arrow) located

far away from the alveolar wall and incorrect positioning of pulmonary venous branches (v) are evident. l, lymphatic

vessels; b, bronchi.

Ten years ago, prof. dr hab. Paweł Stankiewicz and his laboratory in the Department of

Molecular and Human Genetics, Baylor College of Medicine (BCM) in Houston (Texas, USA)

reported the identification of several heterozygous point mutations and deletions of the FOXF1

gene, whose segregation with ACDMPV suggested that changes in the FOXF1 structure or the

expression of its gene are responsible for this disease (Stankiewicz et al. 2009). FOXF1 (fork-head


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box factor1; MIM # 601089) is an evolutionarily conserved transcription factor (Pierrou et al.

1994), encoded in human on chromosome 16 in the cytogenetic position of q24.1 and functioning

in the SHH (sonic hedgehog) signaling pathway from epitelium of developing alveoli to

surrounding mesodermal tissues (Mahlapuu et al. 2001). After joining the lab of prof. Stankiewicz,

I worked, among others on gathering further evidence on the participation of FOXF1 variants in

the pathogenesis of ACDMPV, but the main emphasis was placed on the non-coding aspect of

molecular genetics of this disease, in particular on the identification of a lung-specific enhancer of

FOXF1, non-coding RNA functionally associated with FOXF1 and perhaps also with ACDMPV,

and other non-coding genetic elements potentially relevant to the understanding of the etiology of

ACDMPV due to their destabilizing influence on the integrity of the chr16q24.1 region.

4.4.2 Discussion of the achieved results

4.4.2.1 The FOXF1 variants responsible for ACDMPV

The number of heterozygous point mutations found by us in FOXF1 (pathogenic single-

nucleotide variants, SNV) and one- or several-nucleotide deletions and insertions segregating with

ACDMPV reaches 72. The updated by new variants compilation of known FOXF1 mutations is

shown in Fig. 2. Most of these variants maps in the part of the protein responsible for its interaction

Fig. 2. Compilation of mutations in FOXF1 causative for ACDMPV. The diagram represents cDNA (1137 nt) and

protein (379 aa) of FOXF1. Their 5 'and NH2 ends are on the left.

with the DNA (fork-head domain), while the least on its NH2 end (Pradham et al. 2019). In addition

to the mutations in FOXF1, we also identified 29 ACDMPV cases with large heterozygous

deletions including FOXF1 and one with a deletion limited to its promoter (Fig. 3) (4.2: refs 5,8).

The fact that in 75% (n>150) published cases of ACDMPV structural variants were found in

FOXF1 points to FOXF1 as the main gene causally linked to ACDMPV. In the remaining 25% of


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cases of this disease, the pathogenic variant is located either in the non-coding sequence that

regulates the expression of FOXF1, such as a promoter, enhancer, splicing site or non-coding

RNA, or is associated with a protein gene other than FOXF1.

4.4.2.2 Identification of FOXF1 transcriptional enhancers

Enhancer is a cis-regulatory (to a lesser extent trans-regulatory) DNA sequence, arbitrarily

oriented towards the regulated gene, determining the tissue specificity of the promoter of such a

gene and located usually at a considerable distance from it. It is because of this distance that the

identification of the enhancer is not straightforward. To locate the FOXF1 enhancer, specific to

early lung development, I used the approach often applied in human genetics, consisting of finding

a deletion of a non-coding region located in cis relative to the gene being examined and segregating

with the disease phenotype, or finding a common region of partially overlapping deletions that

would, as in the cases discussed here, segregate with ACDMPV and map in chr16q24.1 but did

not include FOXF1 coding exons or its promoter. In search for deletions, I used a method of the

comparative genomic hybridization (aCGH) on oligonucleotide 3x720K and 4x180K microarrays

designed for chr16q23.3-q24.1. For hybridization, we used DNA isolated from pulmonary blood

or tissue, in which no mutation was detected in FOXF1. The biggest challenge in these studies was

to find a sufficient number of ACDMPV cases with different size deletions, whose comparison

would allow narrowing the region of their overlap to the part of the chromosome that included the

enhancer. Although thanks to cooperation with the ACD Association, our laboratory has access to

samples from all over the world, this disease is so rare and difficult to diagnose that practically

you can count only on identifying several cases of it annually. Nevertheless, we managed to

accumulate a total of 24 ACDMPV cases not having a mutation in FOXF1, with a deletion on

chr16q24.1 not including FOXF1. Molecular analysis of these deletions allowed for identification

of two FOXF1 regulatory regions with enhancer features: one located within the only intron of

FOXF1 and the second at a distance of 272 kb, in centromeric direction from the 5 'end of the

gene.

4.4.2.2.1 Intragenic enhancer (Szafranski et al. 2013 Hum Mutat, 34:1467-1471)

The FOXF1 intragenic enhancer was identified by finding the ACDMPV case with a small

(0.8 kb) heterozygous deletion completely included within the FOXF1 intron (4.2: ref 7). This

deletion was the only variant I found in the coding and non-coding parts of FOXF1 and the only

deletion found on the entire chromosome 16 in the ACDMPV case under investigation. The

possibility of occurrence of abnormal FOXF1 splicing due to this deletion was excluded

experimentally by constructing in the expression plasmid, pcDNA3, a minigen containing

fragments of the exon 1 and 2 surrounding the entire FOXF1 intron or intron with deletion, and

analyzing its splicing in the fibroblast line, IMR-90, from normally developed fetal lungs.

The presence of the enhancer in the FOXF1 intron was suggested by a bioinformatic analysis

of histone 3 (H3) modification in the region of the discussed deletion, found in IMR-90 cells. In

particular, it showed the presence of increased acetylation of H3K27Ac, typical of the active


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enhancer. In addition, the analysis of the ChIP-Seq database for IMR-90 cells, in terms of the

interaction of the examined DNA region with transcription factors, showed the presence of binding

there CTCF and CEBPB. CTCF participates in the creation of chromatin spatial structure, in

particular topologically associating (TAD) domains, within which the enhancer's interaction with

its promoter follows. CEBPB is another transcription factor often found in complex with

enhancers.

I conducted experimental verification of the enhancer activity of the FOXF1 intron by using

an in vitro reporter assay. For this purpose, I inserted in a promoter-less vector, pGL4.10, in front

of the luc2 reporter gene, a weak FOXF1 promoter, and then in the 5 'position before this promoter

either the enhancer fragment to be tested or the same size a freely chosen genome sequence serving

as a negative control. Luc2 transcription was then measured by RT-qPCR in IMR-90 cells

transfected with the vector bearing the enhancer or control vector without the enhancer. Analysis

of luc2 expression revealed that the FOXF1 intronic region under investigation actually enhances

the FOXF1 promoter activity and in the context of the local chromatin organization is probably

spatially related to the promoter due to exon 1 looping.

4.4.2.2.2 Distal enhancer (Szafranski et al. 2013 Genome Res, 23:23-33; Szafranski et al. 2014 Am

J Med Genet A, 164:2013-2019; Szafranski et al. 2016 Clin Epigenet, 8:112)

I identified the FOXF1 distal enhancer after first finding seven (4.2: ref 8) and then dozen more

deletions (4.2: refs 4-6) on chr16q24.1, segregating with ACDMPV but not including FOXF1.

Comparative analysis of these deletions revealed the presence of a region of their mutual overlap

of about 60 kb (chr16: 86,212,040, 40-86,211,919, hg19), 272 kb from the 5 'end of FOXF1 (Fig.

3). The bioinformatics analysis of H3 modification in this region in IMR-90 cells showed the

presence of K27Ac acetylation and K4Me1 methylation, typical of the active enhancer. In the case

of two deletions for which it was possible to isolate RNA with quality suitable for quantitative

analysis, I measured by RT-qPCR the level of FOXF1 transcript. Compared to RNA controls from

the normal lungs, matched by age with ACDMPV lungs, and RNA from the IMR-90 cell culture,

the level of FOXF1 in the lungs with ACDMPV dysplasia turned out to be reduced by 50-75%,

indicating that the deleted region indeed positively regulates the expression of FOXF1 (4.2: ref.

6).

To test the hypothesis postulating the functioning of the distal enhancer through its spatial

approach, together with the associated transcription factors, to the promoter (with looping the

region between it and the promoter), I used the 4C method (chromosome conformation capture on

chip). This method consists in preparing DNA libraries from tested and control cell lines, in which

the DNA regions, linearly spaced apart but in the cell nucleus spatially close together, are

crosslinked with formaldehyde, then cut with a restriction nuclease and ligated, this time with their

spatial neighbors, enabling their identification by sequencing after their amplification by PCR.

Using C4 libraries constructed in pulmonary cells and control lymphoblasts, I identified two

partially overlapping fragments of the enhancer contacting the RNA II polymerase binding site on

the FOXF1 promoter in pulmonary cells, but not in lymphoblasts.


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The bioinformatics analysis of the distal enhancer region showed the presence of binding sites

for transcription factors and proteins involved in maintaining the spatial chromatin structure,

including, confirmed by the C4 method, looping of the region between the enhancer and the

promoter. One of the factors known to regulate FOXF1 expression that might bind to its enhancer

is GLI2. Using the ChIP-Seq method, we demonstrated that GLI2 actually interacts with this

enhancer at sites containing sequences resembling GLI2 binding motifs (4.2: ref 8).

Fig. 3. Compilation of chr16q24.1 deletions causatively related to ACDMPV. The overlap (60 kb) of deletions that

do not include FOXF1 determines the position of the distal enhancer (DEnh).

I performed the functional verification of the enhancer using an in vitro reporter assay and one

of the GLI2 binding regions (chr16: 86,261,498-86,222,908, hg19). The FOXF1 promoter and the

GLI2-binding enhancer region were cloned in the promoter-less vector pSEAP-Basic. The activity


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of the SEAP alkaline phosphatase reporter gene, encoded on this vector, was measured by the

luminescence method after transforming the pulmonary cell culture with the recombinant pSEAP.

The reporter assay showed an increase in the activity of the cloned FOXF1 promoter in the

presence of the tested fragment of the enhancer, but not in the presence of a genomic fragment

used as negative control, indicating the actual enhancer function of the region being tested.

In further studies of the FOXF1 distal enhancer, we focused on determining its most critical

functional region. The identified 60 kb regulatory element can represent a superenhancer

consisting of subregions of different importance from the point of view of the ACDMPV etiology

and regulation of FOXF1. Narrowing the enhancer to its most important part for ACDMPV

became possible after identifying two deletions, one of which related to typical ACDMPV,

shortened the enhancer by 20 kb from its telomeric end, and the second responsible for the delayed

ACDMPV case narrowed the enhancer from its second end to only 15 kb (chr16: 86.223.601-

86.2253,509; hg19) (4.2: refs 4-6). Based on the H3 modification in the IMR-90 cells, the region

directly adjacent to the 15 kb narrowed enhancer core region, from its telomere side, seems to be

equally important for functioning of the enhancer as a whole. For example, there are identified by

the ChIP-Seq binding sites for CTCF, RAD21, CEBPB, TFAP2 and other factors regulating both

the chromatin spatial structure and those that are directly involved in transcription. For one of

them, TFAP2C, we showed for the first time that it actually regulates the expression of FOXF1.

Decreasing the level of TFAP2C by 90% using siRNA (two different RNA duplexes with an LNA-

type modification) reduced the expression of FOXF1 by half (Fig. 4). This change was specific

and e.g. a comparable reduction in the transcript level of TFAP2A or other genes had no effect on

the expression of FOXF1. The TFAP2C regulation of FOXF1 deserves further attention because

of the data suggesting the activation of TFAP2C expression or function by retinoic acid (Oulad-

Abdelghani et al. 1996). Thus, regulation of FOXF1 by TFAP2C may represent a new link between

signaling pathways of SHH and retinoic acid.

The results of our latest enhancer studies also indicate a functionally important role of the

above-mentioned narrowed enhancer region. We have found two cases of heterozygous enhancer

deletions, characterized by a much milder course of the disease in which the sequencing of the

non-deleted (in trans) enhancer allele revealed the presence of single-nucleotide variants, within

TFAP2 and CTCF binding sites, with low incidence in the population and absent in enhancers in

any of the 13 tested patients with ACDMPV. These variants appear to have enhanced the activity

of the non-deleted enhancer allele by, e.g., modifying the binding of transcription factors in it.

4.4.2.2.3 Enhancer lncRNAs (Szafranski et al. 2013 Genome Res, 23:23-33; Szafranski et al. 2014

Am J Med Genet A, 164:2013-2019; Szafranski et al. 2017 Mamm Genome, 28:275-282)

An interesting feature in many respects of the FOXF1 distal enhancer is the presence of genes

for lncRNAs transcribed in the lung, LINC01082, RP11-805I24.3 and LINC01081 (4.2: refs 6,8).

LncRNAs are classified as non-coding protein, non-ribosomal RNA with a length greater than 200

nt, but at least some of them may be traceably translated into short polypeptides of unknown


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function. The role of lncRNA in the function of the FOXF1 enhancer is now the subject of our

intense research. It seems that they can participate in maintaining the spatial proximity of the

Fig. 4. Regulation of FOXF1 expression by TFAP2C with binding site in the region of the distal enhancer.

enhancer and promoter, or they function as scaffolds for transcription factors or enzymatic

complexes, such as PcG (polycomb-group), involved in chromatin modification in the promoter

region or enhancer.

I have found using RNA interference (RNAi) that at least LINC01081 actually regulates the

expression of FOXF1. Lowering the level of this lncRNA in IMR-90 cells using siRNA caused a

reduction in the level of FOXF1 transcript by 15-20% (4.2: ref 6) but may not reflect the actual

magnitude of FOXF1 regulation by this lncRNA. The length of LINC01081 is as much as 60.7 kb

and, as with most lncRNAs, its more effective quiescence may not be possible until it is known

which of their regions is functionally significant. The other two enhancer lncRNAs, LINC01082

and RP11-805I24.3, are of interest because of their almost complete complementarity (their genes

are opposite oriented and overlap). It is not clear whether their transcription and functions are

mutually inhibitory, and whether and how they regulate the expression of FOXF1.

We also created a mouse model of the lncRNA deletion of the distal enhancer. The synthetic

region of the human FOXF1 enhancer has only one gene encoding lncRNA, Gm26878. The

sequence of this RNA is not conserved between the mouse and the human so that it is not known

which human lncRNA corresponds to Gm26878. Using CRISPR/Cas9 technology, we deleted the

entire Gm26878 gene from the mouse genome, thereby eliminating the expression of its lncRNA

in homozygous mice (4.2: ref. 3). However, in contrast to LINC01081, in response to the

elimination of Gm26878, there was no change in Foxf1 expression indicating the existence of


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significant differences in the functioning of the distal enhancer in mice and humans. Nevertheless,

Gm26878 deletion caused a 30% increase in the mortality of mouse embryos.

4.4.2.2.4 Other non-coding RNAs associated with FOXF1: FENDRR (Szafranski et al. 2013

Genome Res, 23:23-33)

In addition to enhancer lncRNA, another lncRNA, FENDRR, not transcribed from the distal

enhancer region, is also associated with FOXF1 and possibly even with ACDMPV. The gene of

this lncRNA lies close to FOXF1 but in the opposite orientation. It is particularly interesting that

its expression is regulated by the same two-way promoter controlling the expression of FOXF1.

The level of FENDRR in the lungs of humans and mice is relatively high (comparable even with

FOXF1), which may suggest that it play a role in their development. Using siRNA, we reduced

the FENDRR level in IMR-90 cells to 85%, which, however, did not significantly affect the FOXF1

expression. On the other hand, reducing by the same method FOXF1 expression by 90% resulted

in FENDRR transcription dropping by half. Therefore, we can conclude that FENDRR is a

mediator of the FOXF1 function, but it remains to be clarified to what extent and if the fall of

FENDRR level is responsible for ACDMPV at all.

The mechanism of FENDRR expression regulation by FOXF1 is currently unknown. The bi-

directional FOXF1 and FENDRR promoter has no binding sites for FOXF1 and is therefore

unlikely to be regulated by FOXF1. It is possible, however, that the intragenic enhancer of FOXF1

also acts as a FENDRR enhancer, interacting with the same two-way promoter, and that the non-

coding, e.g., intronic portion of the FOXF1 transcript contributes to the function of this enhancer.

4.4.2.2.5 The epigenetic aspect of the FOXF1 distal enhancer (Szafranski et al. 2016 Clin Epigenet,

8:112; Szafranski et al. 2016 Hum Genet 135:569-586; Schulze, Szafranski et al. 2019 Clin

Epigenet, 11:60)

Returning to the FOXF1 distal enhancer, it deserves attention in epigenetic terms as well. For

all but one of the pathogenic deletions that included this enhancer, we established the parental

origin of the chromosome 16 on which the patient had a deletion, using the method of analysis of

segregation of parental SNVs in the patient's genome. It turned out that all but one of the deletions

causing ACDMPV occurred on the chromosome 16 inherited from the mother. The model we have

proposed assumes that the enhancer allele on the chromosome inherited from the mother has lower

activity than that on the chromosome from the father. According to this model, the deletion of the

paternal enhancer would be lethal to the fetus, whereas deletion of the maternal enhancer allele

would allow the ACDMPV to develop and be lethal to the newborn (4.2: ref.5). A mechanistic

explanation of differences in the strength of the enhancer may assume the existence of differences

in CpG methylation on maternal and paternal chr16q24.1 or differential H3 modification. Using

the Methyl-Seq method, we performed a DNA methylation analysis of chromosome 16 in two

patients with a heterozygous deletion comprising the FOXF1 distal enhancer (4.2: ref. 1). This

procedure involves the preparation of DNA libraries after treatment with sodium bisulfite. The

bisulfite deaminates unmodified cytosine to uracil, but does not react with 5-methylcytosine. The


14

DNA treated with bisulfite is then amplified by PCR, during which 5-methylcytosine is amplified

as cytosine, and uracil as thymine and then sequenced. This analysis showed increased CpG

methylation in the region of chr16: 86,220,034-86,220,049 (hg19), located merely 11 bp from the

binding site of the lung-specific LUN1 transcription factor. This site can be a regulatory region for

LINC01082 or interact (in a complex with LUN1) with the FOXF1 promoter. I also performed an

independent methylation analysis of enhancer regions characterized by an increased presence of

CpGs, such as, for example, GLI2 binding sites, which due to limited resolution of Methyl-Seq

could be overlooked by this method. I found, by Sanger sequencing of the DNA previously treated

with bisulfite, the existence of differences between the degree of methylation of cytosines on the

maternal and paternal allele of the analyzed GLI2 binding region. However, due to the lack of

informative SNVs in this region, it was not possible to determine which of the alleles (maternal or

paternal) is methylated more. Using an in vitro reporter assay, I showed that differences in the

methylation of this site caused differences in the GLI2 ability to activate the FOXF1 promoter

(4.2: ref. 8). It seems that increased methylation reduces the ability of GLI2 to bind to DNA.

4.4.2.3 Identification of genomic elements responsible for the increased structural instability of the

FOXF1 enhancer (Szafranski et al. 2018 Human Mutation, 39:1916-1925)

The last aspect of the non-coding genetics of FOXF1 and ACDMPV that has been addressed

in the presented work concerns the mechanisms of the FOXF1 enhancer deletions. By aCGH, long-

range PCR and sequencing we have identified almost all the breakpoints of the deletions of both

enhancers. We have found that not only the majority of breakpoints of these deletions map in

repetitive elements LINE1 and SINE Alu, but in the case of distal enhancer, as many as six

breakpoints are mapped to the same L1PA2, six others to very closely located L1PA3 and three to

Alu repeats lying between the two L1PAs. Both L1PAs and neighboring Alus create therefore a

genomic site on chr16q24.1 with reduced stability. The breakpoints of the centromeric end of the

deletions are for five cases in the same L1HS and thus the position of this L1 also determines the

site of reduced genomic stability this time on chr16q23.3. The high degree of similarity of L1HS

and L1PA2 or L1PA3 sequences, exceeding 95%, and the presence of long microhomologies at

the deletion junctions is suggestive of NAHR as a mechanism of deletions.

The history of the appearance of L1PA retrotransposons in chr16q24.1 is also noteworthy. The

phylogenetic analysis of L1PA in this region suggests that they appeared in the line leading to

human, chimpanzee and gorilla, after its separation from the line leading to the orangutan, which

most likely took place 7-12 million years ago. The youngest acquisition seems to be L1PA3, the

presence of which in the region of the FOXF1 enhancer showed only in human and chimpanzee.

4.4.3 Summary of the most important achievements of the habilitant in the presented

publication cycle

- Identification of FOXF1 enhancers on chromosome 16 with the proposed model of their

functioning

- identification of the intragenic enhancer


15

- identification of a distal lung-specific enhancer and evidence of its direct interaction with

the promoter

- Identification of DNA elements responsible for the increased genomic instability in the area of

the FOXF1 distal enhancer and proposed mechanism responsible for deletions in this region

- Identifying LINC01081 as an enhancer lncRNA that regulates the expression of FOXF1, and

identifying FENDRR as such an lncRNA whose expression is regulated by FOXF1

- Characterization of CpG methylation on chromosome 16 including FOXF1 enhancer regions

and proposing a model of epigenetic regulation of FOXF1 expression explaining differences in

ACDMPV presentation

4.4.4 Discussing the possible use of the results achieved

The results of the presented research may find practical use in the prenatal diagnosis of

ACDMPV. Knowledge of the location of the distal FOXF1 enhancer allows, for example, to

automate the identification of the deletion of this enhancer. This is important because about 20%

of ACDMPV cases have deletions that include this enhancer.

Regarding the therapeutic use of the results achieved, it would appear that the most promising

strategy would be to use modified antisense oligos (ASO), complementary to such lncRNA, which

is known to act as a FOXF1 suppressor or as a suppressor of another lncRNA positively regulating

the expression of FOXF1. The lncRNAs, which in this respect would merit a more accurate

examination, include antisense LINC01082 and RP11-805I24.3 encoded within the distal

enhancer.

Supplementary literature

Mahlapuu M et al. (2001) Haploinsufficiency of the forkhead gene Foxf1, a target for sonic hedgehog signaling, causes

lung and foregut malformations. Development, 128:2397-2406.

Oulad-Abdelghani M et al. (1996) AP-2.2: a novel AP-2-related transcription factor induced by retinoic acid during

differentiation of P19 embryonal carcinoma cells. Exp Cell Res, 225:338-347.

Pierrou S et al. (1994) Cloning and characterization of seven human forkhead proteins: binding site specificity and

DNA bending. EMBO J, 13:5002–5012.

Pradhan A et al. (2019) The S52F FOXF1 mutation inhibits STAT3 signaling and causes alveolar capillary dysplasia.

Am J Respir Crit Care Med, 199 [in press]

Slot E et al. (2018) Alveolar capillary dysplasia with misalignment of the pulmonary veins: clinical, histological, and

genetic aspects. Pulm Circ 8:2045894018795143.

Stankiewicz P et al. (2009) Genomic and genic deletions of the FOX gene cluster on 16q24.1 and inactivating

mutations of FOXF1 cause alveolar capillary dysplasia and other malformations. Am J Hum Genet, 84:780-791.

5. OTHER SCIENTIFIC ACHIEVEMENTS OF THE HABILITANT

5.1 List of publications in journals in the Journal Citation Reports database obtained after

a doctorate, not included in the achievement mentioned in point 4

(-) Vincent M, Karolak JA, Deutsch G, Gambin T, Popek E, Isidor B, Szafranski P, Le Caignec

C, Stankiewicz P (2019) Clinical, histopathological, and molecular diagnostics in lethal lung

developmental disorders. Am J Respir Crit Care Med, 199 [in press]


16

IF2017=15.239, MNiSW=50, the number of citations=n/a

(1) Salehi Karlslätt K, Pettersson M, Jäntti N, Szafranski P, Wester T, Husberg B, Ullberg U,

Stankiewicz P, Nordgren A, Lundin J, Lindstrand A, Nordenskjöld A (2019) Rare copy number

variants contribute pathogenic alleles in patients with intestinal malrotation. Mol Genet Genomic

Med, 7(3):e549. doi: 10.1002/mgg3.549. PMID: 30632303

IF2017=2.695, MNiSW=25, the number of citations=0

(2) Karolak JA, Vincent M, Deutsch G, Gambin T, Cogné B, Pichon O, Vetrini F, Mefford HC,

Dines JN, Dishop M, Mowat D, Bennetts B, Gifford AJ, Weber MA, Lee AF, Boerkoel CF, Ward-

Melver C, Besnard T, Petit F, Iben Bache I, Tümer Z, Joubert M, Denis M, Martinovic J, Bénéteau

C, Molin A, Carles D, André G, Bieth E, Chassaing N, Devisme L, Chalabreysse L, Pasquier L,

Secq V, Don M, Orsaria M, Missirian C, Mortreux J, Sanlaville D, Pons L, Küry S, Bézieau S,

Liet J-M, Joram N, Bihouée T, Scott DA, Brown CW, Scaglia F, Tsai AC-H, Grange DK, Phillips

JA3rd, Pfotenhauer JP, Jhangiani SN, Gonzaga CG, Chung WK, Schauer GM, Bartell TM, Mark

H. Lipson MH, Mercer C, van Haeringen A, Liu Q, Popek E, Akdemir ZHC, Lupski JR,

Szafranski P, Isidor B, Le Caignec C, Stankiewicz P (2019) Complex compound inheritance of

lethal lung developmental disorders due to disruption of the TBX-FGF pathway. Am J Hum Genet,

104(2):213-228. doi: 10.1016/j.ajhg.2018.12.010. PMID: 30639323


(3) Towe CT, White FV, Grady RM, Sweet SC, Eghtesady P, Wegner DJ, Sen P, Szafranski P,

Stankiewicz P, Hamvas A, Sessions Cole F, Wambach JA (2018) Infants with atypical

presentations of alveolar capillary dysplasia with misalignment of the pulmonary veins who

underwent bilateral lung transplantation. J Pediatr, 194:158-164.e1. doi:

10.1016/j.jpeds.2017.10.026. PMID: 29198536


(4) Stankiewicz P, Khan TN, Szafranski P, Slattery L, Streff H, Vetrini F, Bernstein JA, Brown

CW, Rosenfeld JA, Rednam S, Scollon S, Bergstrom KL, Parsons DW, Plon SE, Vieira MW,

Quaio CRDC, Baratela WAR, Acosta Guio JC, Armstrong R, Mehta SG, Rump P, Pfundt R,

Lewandowski R, Fernandes EM, Shinde DN, Tang S, Hoyer J, Zweier C, Reis A, Bacino CA,

Xiao R, Breman AM, Smith JL; Deciphering Developmental Disorders Study, Katsanis N,

Bostwick B, Popp B, Davis EE, Yang Y (2017) Haploinsufficiency of the chromatin remodeler

BPTF causes syndromic developmental and speech delay, postnatal microcephaly, and

dysmorphic features. Am J Hum Genet, 101(4):503-515. doi: 10.1016/j.ajhg.2017.08.014. PMID:

28942966



17

(5) Szafranski P (2017) Intercompartmental piecewise gene transfer. Genes (Basel), 8(10). pii:

E260. doi: 10.3390/genes8100260. Review. PMID: 28984842


(6) Zhang J, Gambin T, Yuan B, Szafranski P, Rosenfeld JA, Balwi MA, Alswaid A, Al-Gazali

L, Shamsi AM, Komara M, Ali BR, Roeder E, McAuley L, Roy DS, Manchester DK, Magoulas

P, King LE, Hannig V, Bonneau D, Denommé-Pichon AS, Charif M, Besnard T, Bézieau S, Cogné

B, Andrieux J, Zhu W, He W, Vetrini F, Ward PA, Cheung SW, Bi W, Eng CM, Lupski JR, Yang

Y, Patel A, Lalani SR, Xia F, Stankiewicz P (2017) Haploinsufficiency of the E3 ubiquitin-protein

ligase gene TRIP12 causes intellectual disability with or without autism spectrum disorders, speech

delay, and dysmorphic features. Hum Genet, 136(4):377-386. doi: 10.1007/s00439-017-1763-1.

PMID: 28251352


(7) Szafranski P (2017) Evolutionarily recent, insertional fission of mitochondrial cox2 into

complementary genes in bilaterian Metazoa. BMC Genomics, 18(1):269. doi: 10.1186/s12864-

017-3626-5. PMID: 28359330


(8) Dharmadhikari AV, Sun JJ, Gogolewski K, Carofino BL, Ustiyan V, Hill M, Majewski T,

Szafranski P, Justice MJ, Ray RS, Dickinson ME, Kalinichenko VV, Gambin A, Stankiewicz P

(2016) Lethal lung hypoplasia and vascular defects in mice with conditional Foxf1 overexpression.

Biol Open, 5(11):1595-1606. doi: 10.1242/bio.019208. PMID: 27638768


(9) Gu S, Szafranski P, Akdemir ZC, Yuan B, Cooper ML, Magriñá MA, Bacino CA, Lalani SR,

Breman AM, Smith JL, Patel A, Song RH, Bi W, Cheung SW, Carvalho CM, Stankiewicz P,

Lupski JR (2016) Mechanisms for complex chromosomal insertions. PLoS Genet,

12(11):e1006446. doi: 10.1371/journal.pgen.1006446. PMID: 27880765


(10) Szafranski P, Coban-Akdemir ZH, Rupps R, Grazioli S, Wensley D, Jhangiani SN, Popek

E, Lee AF, Lupski JR, Boerkoel CF, Stankiewicz P (2016) Phenotypic expansion of TBX4

mutations to include acinar dysplasia of the lungs. Am J Med Genet A, 170(9):2440-2444. doi:

10.1002/ajmg.a.37822. PMID: 27374786


(11) Reiter J, Szafranski P, Breuer O, Perles Z, Dagan T, Stankiewicz P, Kerem E (2016) Variable

phenotypic presentation of a novel FOXF1 missense mutation in a single family. Pediatr

Pulmonol, 51(9):921-927. doi: 10.1002/ppul.23425. PMID: 27145217


18


(12) Prothro SL, Plosa E, Markham M, Szafranski P, Stankiewicz P, Killen SA (2016) Prenatal

diagnosis of alveolar capillary dysplasia with misalignment of pulmonary veins. J Pediatr,

170:317-318. doi: 10.1016/j.jpeds.2015.11.041. PMID: 26703872


(13) Macias A, Gambin T, Szafranski P, Jhangiani SN, Kolasa A, Obersztyn E, Lupski JR,

Stankiewicz P, Kaminska A (2016) CAV3 mutation in a patient with transient hyperCKemia and

myalgia. Neurol Neurochir Pol, 50(6):468-473. doi: 10.1016/j.pjnns.2016.06.008. PMID:

27772553


(14) Smyk M, Roeder E, Cheung SW, Szafranski P, Stankiewicz P (2015) A de novo 1.58 Mb

deletion, including MAP2K6 and mapping 1.28 Mb upstream to SOX9, identified in a patient with

Pierre Robin sequence and osteopenia with multiple fractures. Am J Med Genet A, 167A(8):1842-

1850. doi: 10.1002/ajmg.a.37057. PMID: 26059046


(15) Szafranski P, Golla S, Jin W, Fang P, Hixson P, Matalon R, Kinney D, Bock HG, Craigen

W, Smith JL, Bi W, Patel A, Wai Cheung S, Bacino CA, Stankiewicz P (2015)

Neurodevelopmental and neurobehavioral characteristics in males and females with CDKL5

duplications. Eur J Hum Genet, 23(7):915-921. doi: 10.1038/ejhg.2014.217. PMID: 25315662


(16) Startek M*, Szafranski P*, Gambin T, Campbell IM, Hixson P, Shaw CA, Stankiewicz P,

Gambin A (2015) Genome-wide analyses of LINE-LINE-mediated nonallelic homologous

recombination. Nucleic Acids Res, 43(4):2188-2198. doi: 10.1093/nar/gku1394. PMID: 25613453

*Equal contribution

The publication has been highlighted as a breakthrough paper


(17) Szafranski P, Von Allmen GK, Graham BH, Wilfong AA, Kang SH, Ferreira JA, Upton SJ,

Moeschler JB, Bi W, Rosenfeld JA, Shaffer LG, Wai Cheung S, Stankiewicz P, Lalani SR (2015)

6q22.1 microdeletion and susceptibility to pediatric epilepsy. Eur J Hum Genet, 23(2):173-179.

doi: 10.1038/ejhg.2014.75. PMID: 24824130



19

(18) Dharmadhikari AV, Szafranski P, Kalinichenko VV, Stankiewicz P (2015) Genomic and

epigenetic complexity of the FOXF1 locus in 16q24.1: Implications for development and disease.

Curr Genomics, 16(2):107-116. doi: 10.2174/1389202916666150122223252. PMID: 26085809


(19) Dharmadhikari AV, Gambin T, Szafranski P, Cao W, Probst FJ, Jin W, Fang P, Gogolewski

K, Gambin A, George-Abraham JK, Golla S, Boidein F, Duban-Bedu B, Delobel B, Andrieux J,

Becker K, Holinski-Feder E, Cheung SW, Stankiewicz P (2014) Molecular and clinical analyses

of 16q24.1 duplications involving FOXF1 identify an evolutionarily unstable large minisatellite.

BMC Med Genet, 15:128. doi: 10.1186/s12881-014-0128-z. PMID: 25472632


(20) Campbell IM, Yuan B, Robberecht C, Pfundt R, Szafranski P, McEntagart ME, Nagamani

SC, Erez A, Bartnik M, Wiśniowiecka-Kowalnik B, Plunkett KS, Pursley AN, Kang SH, Bi W,

Lalani SR, Bacino CA, Vast M, Marks K, Patton M, Olofsson P, Patel A, Veltman JA, Cheung

SW, Shaw CA, Vissers LE, Vermeesch JR, Lupski JR, Stankiewicz P (2014) Parental somatic

mosaicism is underrecognized and influences recurrence risk of genomic disorders. Am J Hum

Genet, 95(2):173-182. doi: 10.1016/j.ajhg.2014.07.003. PMID: 25087610


(21) Sen P, Dharmadhikari AV, Majewski T, Mohammad MA, Kalin TV, Zabielska J, Ren X,

Bray M, Brown HM, Welty S, Thevananther S, Langston C, Szafranski P, Justice MJ,

Kalinichenko VV, Gambin A, Belmont J, Stankiewicz P (2014) Comparative analyses of lung

transcriptomes in patients with alveolar capillary dysplasia with misalignment of pulmonary veins

and in Foxf1 heterozygous knockout mice. PLoS One, 9(4):e94390. doi:

10.1371/journal.pone.0094390. PMID: 24722050


(22) Smyk M, Szafranski P, Startek M, Gambin A, Stankiewicz P (2013) Chromosome

conformation capture-on-chip analysis of long-range cis-interactions of the SOX9 promoter.

Chromosome Res, 21(8):781-788. doi: 10.1007/s10577-013-9386-4. PMID: 24254229


(23) Witsch J, Szafranski P, Chen CA, Immken L, Simpson Patel G, Hixson P, Cheung SW,

Stankiewicz P, Schaaf CP (2013) Intragenic deletions of the IGF1 receptor gene in five individuals

with psychiatric phenotypes and developmental delay. Eur J Hum Genet, 21(11):1304-1307. doi:

10.1038/ejhg.2013.42. PMID: 23486542



20

(24) Dittwald P, Gambin T, Szafranski P, Li J, Amato S, Divon MY, Rodríguez Rojas LX, Elton

LE, Scott DA, Schaaf CP, Torres-Martinez W, Stevens AK, Rosenfeld JA, Agadi S, Francis D,

Kang SH, Breman A, Lalani SR, Bacino CA, Bi W, Milosavljevic A, Beaudet AL, Patel A, Shaw

CA, Lupski JR, Gambin A, Cheung SW, Stankiewicz P (2013) NAHR-mediated copy-number

variants in a clinical population: mechanistic insights into both genomic disorders and Mendelizing

traits. Genome Res, 23(9):1395-1409. doi: 10.1101/gr.152454.112. PMID: 23657883


(25) Sen P, Yang Y, Navarro C, Silva I, Szafranski P, Kolodziejska KE, Dharmadhikari AV,

Mostafa H, Kozakewich H, Kearney D, Cahill JB, Whitt M, Bilic M, Margraf L, Charles A,

Goldblatt J, Gibson K, Lantz PE, Garvin AJ, Petty J, Kiblawi Z, Zuppan C, McConkie-Rosell A,

McDonald MT, Peterson-Carmichael SL, Gaede JT, Shivanna B, Schady D, Friedlich PS, Hays

SR, Palafoll IV, Siebers-Renelt U, Bohring A, Finn LS, Siebert JR, Galambos C, Nguyen L, Riley

M, Chassaing N, Vigouroux A, Rocha G, Fernandes S, Brumbaugh J, Roberts K, Ho-Ming L, Lo

IF, Lam S, Gerychova R, Jezova M, Valaskova I, Fellmann F, Afshar K, Giannoni E, Muhlethaler

V, Liang J, Beckmann JS, Lioy J, Deshmukh H, Srinivasan L, Swarr DT, Sloman M, Shaw-Smith

C, van Loon RL, Hagman C, Sznajer Y, Barrea C, Galant C, Detaille T, Wambach JA, Cole FS,

Hamvas A, Prince LS, Diderich KE, Brooks AS, Verdijk RM, Ravindranathan H, Sugo E, Mowat

D, Baker ML, Langston C, Welty S, Stankiewicz P (2013) Novel FOXF1 mutations in sporadic

and familial cases of alveolar capillary dysplasia with misaligned pulmonary veins imply a role

for its DNA binding domain. Hum Mutat, 34(6):801-811. doi: 10.1002/humu.22313. PMID:

23505205

IF2013=5.122 MNiSW=40, the number of citations=47

(26) Lalani SR, Shaw C, Wang X, Patel A, Patterson LW, Kołodziejska K, Szafranski P, Ou Z,

Tian Q, Kang SH, Jinnah A, Ali S, Malik A, Hixson P, Potocki L, Lupski JR, Stankiewicz P,

Bacino CA, Dawson B, Beaudet AL, Boricha FM, Whittaker R, Li C, Ware SM, Cheung SW,

Penny DJ, Jefferies JL, Belmont JW (2013) Rare DNA copy number variants in cardiovascular

malformations with extracardiac abnormalities. Eur J Hum Genet, 21(2):173-181. doi:

10.1038/ejhg.2012.155. PMID: 22929023


(27) Dharmadhikari AV, Kang SH, Szafranski P, Person RE, Sampath S, Prakash SK, Bader PI,

Phillips JA 3rd, Hannig V, Williams M, Vinson SS, Wilfong AA, Reimschisel TE, Craigen WJ,

Patel A, Bi W, Lupski JR, Belmont J, Cheung SW, Stankiewicz P (2012) Small rare recurrent

deletions and reciprocal duplications in 2q21.1, including brain-specific ARHGEF4 and GPR148.

Hum Mol Genet, 21(15):3345-3355. doi: 10.1093/hmg/dds166. PMID: 22543972



21

(28) Ou Z, Stankiewicz P, Xia Z, Breman AM, Dawson B, Wiszniewska J, Szafranski P, Cooper

ML, Rao M, Shao L, South ST, Coleman K, Fernhoff PM, Deray MJ, Rosengren S, Roeder ER,

Enciso VB, Chinault AC, Patel A, Kang SH, Shaw CA, Lupski JR, Cheung SW (2011)

Observation and prediction of recurrent human translocations mediated by NAHR between

nonhomologous chromosomes. Genome Res, 21(1):33-46. doi: 10.1101/gr.111609.110. PMID:

21205869


(29) Ramocki MB*, Bartnik M*, Szafranski P*, Kołodziejska KE, Xia Z, Bravo J, Miller GS,

Rodriguez DL, Williams CA, Bader PI, Szczepanik E, Mazurczak T, Antczak-Marach D, Coldwell

JG, Akman CI, McAlmon K, Cohen MP, McGrath J, Roeder E, Mueller J, Kang SH, Bacino CA,

Patel A, Bocian E, Shaw CA, Cheung SW, Mazurczak T, Stankiewicz P (2010) Recurrent distal

7q11.23 deletion including HIP1 and YWHAG identified in patients with intellectual disabilities,

epilepsy, and neurobehavioral problems. Am J Hum Genet, 87(6):857-865. doi:

10.1016/j.ajhg.2010.10.019. PMID: 21109226

*Equal contribution


(30) Szafranski P*, Schaaf CP*, Person RE, Gibson IB, Xia Z, Mahadevan S, Wiszniewska J,

Bacino CA, Lalani S, Potocki L, Kang SH, Patel A, Cheung SW, Probst FJ, Graham BH, Shinawi

M, Beaudet AL, Stankiewicz P (2010) Structures and molecular mechanisms for common 15q13.3

microduplications involving CHRNA7: benign or pathological? Hum Mutat, 31(7):840-850. doi:

10.1002/humu.21284. PMID: 20506139


*Equal contribution

(31) Szafranski P (2009) The mitochondrial trn-cox1 locus: rapid evolution in Pompilidae and

evidence of bias in cox1 initiation and termination codon usage. Mitochondrial DNA, 20(1):15-25.

PMID: 19565676


(32) Zhao M, Szafranski P, Hall CA, Goode S (2008) Basolateral junctions utilize warts signaling

to control epithelial-mesenchymal transition and proliferation crucial for migration and invasion

of Drosophila ovarian epithelial cells. Genetics, 178(4):1947-1971. doi:

10.1534/genetics.108.086983. PMID: 18430928


(33) Wu L, Yong SL, Fan C, Ni Y, Yoo S, Zhang T, Zhang X, Obejero-Paz CA, Rho HJ, Ke T,

Szafranski P, Jones SW, Chen Q, Wang QK (2008) Identification of a new co-factor, MOG1,


22

required for the full function of cardiac sodium channel Nav 1.5. J Biol Chem, 283(11):6968-6978.

doi: 10.1074/jbc.M709721200. PMID: 18184654


(34) Szafranski P, Goode S (2007) Basolateral junctions are sufficient to suppress epithelial

invasion during Drosophila oogenesis. Dev Dyn, 236(2):364-373. PMID: 17103414


(35) Szafranski P, Goode S (2004) A Fasciclin 2 morphogenetic switch organizes epithelial cell

cluster polarity and motility. Development, 131(9):2023-2036. PMID: 15056617


(36) Tian XL, Kadaba R, You SA, Liu M, Timur AA, Yang L, Chen Q, Szafranski P, Rao S, Wu

L, Housman DE, DiCorleto PE, Driscoll DJ, Borrow J, Wang Q (2004) Identification of an

angiogenic factor that when mutated causes susceptibility to Klippel-Trenaunay syndrome.

Nature. 2004; 427(6975):640-645. PMID: 14961121

The paper is highlighted in News & Views, Nature 427, pp. 592-594, in Faculty of 1000 Biology,

and PR Newswire.


(37) Chen S, Zhang L, Bryant RM, Vincent GM, Flippin M, Lee JC, Brown E, Zimmerman F,

Rozich R, Szafranski P, Oberti C, Sterba R, Marangi D, Tchou PJ, Chung MK, Wang Q (2003)

KCNQ1 mutations in patients with a family history of lethal cardiac arrhythmias and sudden death.

Clin Genet, 63(4):273-282. PMID: 12702160


(38) Huang JH, Rajkovic A, Szafranski P, Ochsner S, Richards J, Goode S (2003) Expression of

Drosophila neoplastic tumor suppressor genes discslarge, scribble, and lethal giant larvae in the

mammalian ovary. Gene Expr Patterns, 3(1):3-11. PMID: 12609595


(39) Szafranski P (2002) New host plant and distributional records for some Eburia Lepeletier &

Audinet-Serville (Coleoptera: Cerambycidae) in North America including Mexico. Pan-Pacific

Entomol, 78(1):66-67.


(40) Wang Q, Timur AA, Szafranski P, Sadgephour A, Jurecic V, Cowell J, Baldini A, Driscoll

DJ (2001) Identification and molecular characterization of de novo translocation

t(8;14)(q22.3;q13) associated with a vascular and tissue overgrowth syndrome. Cytogenet Cell

Genet, 95(3-4):183-188. PMID: 12063397


23


(41) Szafranski P, Smith CL, Cantor CR (1997) Principal transcription sigma factors of

Pseudomonas putida strains mt-2 and G1 are significantly different. Gene, 204(1-2):133-138.

PMID: 9434175


(42) Szafranski P, Smith CL, Cantor CR (1997) Cloning and analysis of the dnaG gene encoding

Pseudomonas putida DNA primase. Biochim Biophys Acta, 1352(3):243-248. PMID: 9224947


(43) Szafranski P, Mello CM, Sano T, Smith CL, Kaplan DL, Cantor CR (1997) A new approach

for containment of microorganisms: dual control of streptavidin expression by antisense RNA and

the T7 transcription system. Proc Natl Acad Sci U S A, 94(4):1059-1063. PMID: 9037005


(44) Yaar R, Szafranski P, Cantor CR, Smith CL (1996) In situ detection of tandem DNA repeat

length. Genet Anal, 13(5):113-118. PMID: 9021399

IF1997=0.696, MNiSW=n/a, the number of citations=2

(45) Szafranski P (1994) Somatic mosaicism in Maniola jurtina (Nymphalidae: Satyridae). J

Lepid Soc, 48(3):264-265.


(46) Szafranski P (1992) On the evolution of the bacterial major sigma factors. J Mol Evol,

34(5):465-467. PMID: 1602496


(47) Szafranski P (1992) New host record for Cassida ferruginea Goeze (Coleoptera:

Chrysomelidae). Coleopterists Bull, 46(1):103.


(48) Szafranski P, Smagowicz WJ (1992) Relative affinities of nucleotide substrates for the yeast

tRNA gene transcription complex. Z Naturforsch C, 47(3-4):320-321. PMID: 1590892


(49) Szafranski P, Smagowicz WJ (1991) Role of metal ions in the assembly and decay of the

transcription initiation complex on tRNA gene in yeast extracts. FEBS Lett, 293(1-2):42-44.

PMID: 1959669



24

(50) Szafranski P, Godson GN (1990) Hypersensitive mung bean nuclease cleavage sites in

Plasmodium knowlesi DNA. Gene, 88(2):141-147. PMID: 2140809


5.2 List of other publications obtained after doctoral studies

The list of other publications in scientific journals, chapters in books, and conference materials is

included in attachment no. 5.

5.3 List of patents

(1) Szafranski P, Mello CM, Sano T, Smith CL, Kaplan DL, Cantor CR. Compositions and

methods for controlling genetically engineered organisms; US Patent No. 6,287,844 issued

09.11.2001

(2) Smith CL, Yaar R, Szafranski P, Cantor CR. Nucleic acid detection methods; US Patent No.

5,753,439 issued 05.19.1998

(3) Szafranski P, Mello CM, Sano T, Marx KA, Cantor CR, Kaplan DL, Smith CL. Biotin-binding

containment systems; US Patent No. 5,679,533 issued 10.21.1997

5.4 Discussion of other scientific and research achievements

5.4.1 Introduction

The works comprising the doctoral dissertation and part of my later research concerned the

broadly understood regulation of gene expression with particular emphasis on the initiation of

transcription. In the dissertation, the major focus is on the recognition of the mechanism of binding

of nucleotide substrates in the catalytic center of DNA dependent bacterial RNA polymerase. After

returning from the internship at NYU Medical Center (New York), where I worked on the

regulation of gene expression in malaria parasite, I continued the IBB PAS transcription initiation

study using RNA polymerase III (PolIII) from S. cerevisiae as a model system (5.1: refs 48 , 49).

We showed, among others further evidence for the participation of TFIIIC (tau) in the formation

of a transcription complex rather than in the RNA polymerization process itself, and we found the

first solid premises for the existence of the second, in addition to the catalytic, stoichiometric

binding site of the Mg2 + ion on the PolIII transcription complex. The scientific achievement

underlying this habilitation application is to some extent also a continuation of those earlier studies

on the initiation of transcription, as it relates to the identification and mechanisms of transcriptional

enhancers. During the work that I later conducted in two different centers in the USA (BU in

Boston and BCM in Houston), I also initiated or continued a number of other research topics, the

most important results of which are summarized below.


25

5.4.2 Molecular genetics of malformations other than ACDMPV

- Identification of the gene and protein of the new human angiogenic factor, AGGF1 (VG5Q),

whose variants are responsible for vascular malformations and KTS (Klippel-Trénaunay-Weber

Syndrome) (5.1: ref. 36)

- Identification of a new cofactor of the sodium channel Nav1.5, mutations of which cause various

types of cardiac arrhythmias (LQTS, Brugada syndrome and others) (5.1.: ref. 33), and

identification of new mutations in the KCNQT potassium channel gene responsible for LQTS (5.1:

ref. 37)

- Identification of the first variant of the TBX4 gene causing acinar dysplasia (5.1: ref. 10) and

studies of the genetic basis of pulmonary malformations other than ACDMPV (5.1: ref. 2)

- Identification of new genes and pathogenic nucleotide variants for neurological and psychiatric

diseases (5.1: refs 4,6,13,15,17,23,27,29,30)

- Proposing new mechanisms of genomic rearrangement (deletions, duplications and

translocations) (5.1: refs 9,16,24,28)

- Establishment of animal (Drosophila) model of human tumor invasion (5.1: refs 32,34,35,38).

This model allows screening of chemical libraries to identify compounds that block collective cell

migration. While working on this project, we have identified a new signaling pathway responsible

for the active migration of cells during normal development and cancerous transformation.

5.4.3 Instability and evolution of mitochondrial genomes (mtDNA) in Metazoa (5.1: refs

5,7,31)

- Discovery of the first divided mitochondrial protein gene, cox2, and new mitochondrial genes in

animal organism (5.1: refs 5,7).

Mitochondrial genes divided into two fragments, functioning as independent genes, were

known only in two small groups of single-celled organisms, not in multicellular animals or plants.

Identifying new genes in mtDNA in the animal body is also interesting because the gene content

in mtDNA of animals is very reduced, usually extremely stable, and the regions between genes are

residual. The function of newly discovered genes is unknown, but one of them may code for a new

type of nuclease.

5.4.4 Pseudomonas putida: replication, transcription, and biotechnology (5.1: refs 42-44)

- Discovery of a new structural motif in DNA primase from P. putida.

My early research on transcription in E. coli was in some sense also continued in the work on

P. putida, which I conducted at the BU in Boston. I identified there, among others, dnaG gene

coding for DNA primase being a simple RNA polymerase synthesizing primers for DNA

replication (5.1: ref. 42). An interesting thread of this work was the identification of a new protein

structure of this primase probably responsible for a new type of regulation of DNA replication in

P. putida. This may be important for the work to find inhibitors of antibiotic-resistant P.

aeruginosa infections in cystic fibrosis.


26

- Creation of a synthetic genetic system for P. putida that enables the safe use of this bacterium to

remove petrochemical pollutants (pkt 5.1: ref.43).

This system was generated as a plasmid construct and functioned by inducing self-destruction

of bacteria after they completed the degradation of a specific hydrocarbon pollution. The results

of these tests have been patented (pkt 5.3: refs 1,3).

5.5 Bibliometric indicators

Total impact factor according to the Journal Citation Reports (JCR) (according to the year of

publication): 288.69

Total number of MniSW points: 1873

The number of article citations according to the Web of Science database (WoS): 1252; without

selfcitations: 1161

Hirsch index according to the Web of Science database: 19

The total number of published scientific articles according to the Web of Science database: 65

5.6 Awards and distinctions for scientific activity

(1) Poster Talk and Best Poster Award at the American Society of Human Genetics (ASHG)

conference in San Diego, California, USA; 2018

(2) Award of the Scientific Secretary of the Polish Academy of Sciences; 1985

(3) NATO-EMBO-FEBS scholarship at the Spetsai International Summer School of Molecular

Biology "Regulation of gene expression in prokaryotes and eukaryotes"; 1982

(4) Graduate student fellowship of the Polish Academy of Sciences; 1978-1982

(5) Distinction of the MA thesis (Faculty of Biology, University of Warsaw); 1978

6. DIDACTICS AND POPULARIZATION OF SCIENCE

6.1 Active participation in scientific conferences

6.1.1 Talks delivered at the invitation of conference organizers

(1) Szafranski P (2018) Interrupted genes in mitochondria: a split cox2 and its expression. 2nd

International Caparica Conference in Splicing. 2018; Caparica, Portugal.

6.1.2 Posters presented at conferences

The list of poster abstracts presented in the last three years is shown in the attachment no. 8.

6.3 Participation in editorial committees and scientific councils of journals

6.3.1. Participation in editorial works

Name of the journal: Genes (ISSN 2073-4425; CODEN: GENEG9)

bibliometric indicators of the journal: IF2017: 3.191, 5-YIF2017: 3.286

publisher's name: MDPI

Period of participation: from 2018


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Character of participation: member of the Editorial Board (editor with the power to decide about

accepting works for printing)

6.3.2 Participation as a reviewer of the publication

List of magazines to which I reviewed articles: Proceedings of the National Academy of Sciences

of the USA, Frontiers in Biology, Current Biotechnology, Clinical Genetics, Journal of Obstetrics

and Gynecology Research, Genes, American Journal of Medical Genetics.

6.4 Membership in scientific societies

(1) American Society for Biochemistry and Molecular Biology: since 2012

(2) American Society of Human Genetics: since 2010

(3) American Association for the Advancement of Science: since 2002

(4) Genetic Society of America: since 2000

6.5 Popularization of science

Szafranski P. Mitochondrial gene discontinuity that translates into fragmented functional

proteins. Atlas of Science. 2018; [pp. 1-3]. http://atlasofscience.org

6.6 Mentoring and training students and graduates

Details of this activity are presented in attachment no. 8.

http://atlasofscience.org/

Documents

AUTOREFERAT Dr Przemyslaw Szafranski‚. 4 Autoreferat wersja ang. P.Sz_..pdfAUTOREFERAT Dr Przemyslaw Szafranski Department of Molecular and Human Genetics, Baylor College of Medicine,