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Gene regulation by different proteins of TGFβ superfamily
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.ORCID-id: 0000-0003-1177-0839
2018 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

The present thesis discusses how gene regulation by transforming growth factor β (TGFβ) family cytokines is affected by post-translational modifications of different transcription factors. The thesis also focuses on gene regulation by transcription factors involved in TGFβ signaling.

The importance of the poly ADP-ribose polymerase (PARP) family in controlling gene expression in response to TGFβ and bone morphogenetic protein (BMP) is analyzed first. PARP2, along with PARP1, ADP-ribosylates Smad2 and Smad3, the signaling mediators of TGFβ. On the other hand, poly ADP-ribose glycohydrolase (PARG) removes the ADP-ribose from Smad2/3 and antagonizes PARP1 and PARP2. ADP-ribosylation of Smads in turn affects their DNA binding capacity. We then illustrate how PARP1 and PARG can regulate gene expression in response to BMP that signals via Smad1, 5. Over-expression of PARP1 suppressed the transcriptional activity of Smad1/5. Knockdown of PARP1 or over-expression of PARG enhanced the transcriptional activity of BMP-Smads on target genes. Hence our data suggest that ADP-ribosylation of Smad proteins controls both TGFβ and BMP signaling. 

I then focus on elucidating novel genes that are regulated by ZEB1 and Snail1, two key transcriptional factors in TGFβ signaling, known for their ability to induce EMT and cancer metastasis. Chromatin immunoprecipitation-sequencing (ChIP-seq) and targeted whole genome transcriptomics in triple negative breast cancer cells were used, to find binding regions and the functional impact of ZEB1 and Snail1 throughout the genome. ZEB1 binds to the regulatory sequences of a wide range of genes, not only related to cell invasion, pointing to new functions of ZEB1. On the other hand, Snail1 regulated only a few genes, especially related to signal transduction and cellular movement. Further functional analysis revealed that ZEB1 could regulate the anchorage-independent growth of the triple negative breast cancer cells, whereas Snail1 could regulate the expression of BMP6 in these cells. We have therefore elucidated novel functional roles of the two transcription factors, Snail1 and ZEB1 in triple negative breast cancer cells.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2018. , s. 50
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1404
Emneord [en]
EMT, Snail1, ZEB1, TGFβ, BMP, Gene regulation
HSV kategori
Identifikatorer
URN: urn:nbn:se:uu:diva-334411ISBN: 978-91-513-0172-3 (tryckt)OAI: oai:DiVA.org:uu-334411DiVA, id: diva2:1159605
Disputas
2018-01-29, Room B42, BMC, Husargatan 3, Uppsala, 09:15 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2017-12-29 Laget: 2017-11-23 Sist oppdatert: 2018-03-08
Delarbeid
1. Fine-Tuning of Smad Protein Function by Poly(ADP-Ribose) Polymerases and Poly(ADP-Ribose) Glycohydrolase during Transforming Growth Factor β Signaling
Åpne denne publikasjonen i ny fane eller vindu >>Fine-Tuning of Smad Protein Function by Poly(ADP-Ribose) Polymerases and Poly(ADP-Ribose) Glycohydrolase during Transforming Growth Factor β Signaling
Vise andre…
2014 (engelsk)Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, nr 8, s. e103651-Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

BACKGROUND:

Initiation, amplitude, duration and termination of transforming growth factor β (TGFβ) signaling via Smad proteins is regulated by post-translational modifications, including phosphorylation, ubiquitination and acetylation. We previously reported that ADP-ribosylation of Smads by poly(ADP-ribose) polymerase 1 (PARP-1) negatively influences Smad-mediated transcription. PARP-1 is known to functionally interact with PARP-2 in the nucleus and the enzyme poly(ADP-ribose) glycohydrolase (PARG) can remove poly(ADP-ribose) chains from target proteins. Here we aimed at analyzing possible cooperation between PARP-1, PARP-2 and PARG in regulation of TGFβ signaling.

METHODS:

A robust cell model of TGFβ signaling, i.e. human HaCaT keratinocytes, was used. Endogenous Smad3 ADP-ribosylation and protein complexes between Smads and PARPs were studied using proximity ligation assays and co-immunoprecipitation assays, which were complemented by in vitro ADP-ribosylation assays using recombinant proteins. Real-time RT-PCR analysis of mRNA levels and promoter-reporter assays provided quantitative analysis of gene expression in response to TGFβ stimulation and after genetic perturbations of PARP-1/-2 and PARG based on RNA interference.

RESULTS:

TGFβ signaling rapidly induces nuclear ADP-ribosylation of Smad3 that coincides with a relative enhancement of nuclear complexes of Smads with PARP-1 and PARP-2. Inversely, PARG interacts with Smads and can de-ADP-ribosylate Smad3 in vitro. PARP-1 and PARP-2 also form complexes with each other, and Smads interact and activate auto-ADP-ribosylation of both PARP-1 and PARP-2. PARP-2, similar to PARP-1, negatively regulates specific TGFβ target genes (fibronectin, Smad7) and Smad transcriptional responses, and PARG positively regulates these genes. Accordingly, inhibition of TGFβ-mediated transcription caused by silencing endogenous PARG expression could be relieved after simultaneous depletion of PARP-1.

CONCLUSION:

Nuclear Smad function is negatively regulated by PARP-1 that is assisted by PARP-2 and positively regulated by PARG during the course of TGFβ signaling.

HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-231920 (URN)10.1371/journal.pone.0103651 (DOI)000341302700014 ()25133494 (PubMedID)
Tilgjengelig fra: 2014-09-11 Laget: 2014-09-11 Sist oppdatert: 2017-12-05bibliografisk kontrollert
2. Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation
Åpne denne publikasjonen i ny fane eller vindu >>Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation
Vise andre…
2016 (engelsk)Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, nr 24, s. 12706-12723Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

We previously established a mechanism of negative regulation of transforming growth factor beta signaling mediated by the nuclear ADP-ribosylating enzyme poly-(ADP-ribose) polymerase 1 (PARP1) and the deribosylating enzyme poly-(ADP-ribose) glycohydrolase (PARG), which dynamically regulate ADP-ribosylation of Smad3 and Smad4, two central signaling proteins of the pathway. Here we demonstrate that the bone morphogenetic protein (BMP) pathway can also be regulated by the opposing actions of PARP1 and PARG. PARG positively contributes to BMP signaling and forms physical complexes with Smad5 and Smad4. The positive role PARG plays during BMP signaling can be neutralized by PARP1, as demonstrated by experiments where PARG and PARP1 are simultaneously silenced. In contrast to PARG, ectopic expression of PARP1 suppresses BMP signaling, whereas silencing of endogenous PARP1 enhances signaling and BMP-induced differentiation. The two major Smad proteins of the BMP pathway, Smad1 and Smad5, interact with PARP1 and can be ADP-ribosylated in vitro, whereas PARG causes deribosylation. The overall outcome of this mode of regulation of BMP signal transduction provides a fine-tuning mechanism based on the two major enzymes that control cellular ADP-ribosylation.

HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-299724 (URN)10.1074/jbc.M116.729699 (DOI)000378119900024 ()27129221 (PubMedID)
Forskningsfinansiär
Swedish Research Council, K2010-67X-14936-07-3 K2013-66X-14936-10-5
Tilgjengelig fra: 2016-07-26 Laget: 2016-07-26 Sist oppdatert: 2018-01-10bibliografisk kontrollert
3. Genome-wide binding of transcription factor ZEB1 in triple-negative breast cancer cells
Åpne denne publikasjonen i ny fane eller vindu >>Genome-wide binding of transcription factor ZEB1 in triple-negative breast cancer cells
2018 (engelsk)Inngår i: Journal of Cellular Physiology, ISSN 0021-9541, E-ISSN 1097-4652, Vol. 233, nr 10, s. 7113-7127Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Zinc finger E-box binding homeobox 1 (ZEB1) is a transcriptional regulator involved in embryonic development and cancer progression. ZEB1 induces epithelial-mesenchymal transition (EMT). Triple-negative human breast cancers express high ZEB1 mRNA levels and exhibit features of EMT. In the human triple-negative breast cancer cell model Hs578T, ZEB1 associates with almost 2,000 genes, representing many cellular functions, including cell polarity regulation (DLG2 and FAT3). By introducing a CRISPR-Cas9-mediated 30bp deletion into the ZEB1 second exon, we observed reduced migratory and anchorage-independent growth capacity of these tumor cells. Transcriptomic analysis of control and ZEB1 knockout cells, revealed 1,372 differentially expressed genes. The TIMP metallopeptidase inhibitor 3 and the teneurin transmembrane protein 2 genes showed increased expression upon loss of ZEB1, possibly mediating pro-tumorigenic actions of ZEB1. This work provides a resource for regulators of cancer progression that function under the transcriptional control of ZEB1. The data confirm that removing a single EMT transcription factor, such as ZEB1, is not sufficient for reverting the triple-negative mesenchymal breast cancer cells into more differentiated, epithelial-like clones, but can reduce tumorigenic potential, suggesting that not all pro-tumorigenic actions of ZEB1 are linked to the EMT.

Emneord
ZEB1, EMT, ChIP-seq, CRISPR-Cas9
HSV kategori
Forskningsprogram
Biokemi; Biologi med inriktning mot molekylär cellbiologi
Identifikatorer
urn:nbn:se:uu:diva-334438 (URN)10.1002/jcp.26634 (DOI)000438352300071 ()29744893 (PubMedID)
Forskningsfinansiär
Swedish Research Council, 2015-02757Swedish Research Council, K2013-66X-14936-10-5Swedish Cancer Society, CAN 2012/438Swedish Cancer Society, CAN 2015/438Swedish Cancer Society, CAN 2016/445
Tilgjengelig fra: 2017-11-23 Laget: 2017-11-23 Sist oppdatert: 2018-09-24bibliografisk kontrollert
4. Genome-wide binding of transcription factor Snail1 in triple-negative breast cancer cells
Åpne denne publikasjonen i ny fane eller vindu >>Genome-wide binding of transcription factor Snail1 in triple-negative breast cancer cells
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
Emneord
ChIP-Seq, Snail1, Ampliseq, EMtT, BMP6
HSV kategori
Forskningsprogram
Biokemi; Biologi med inriktning mot molekylär cellbiologi
Identifikatorer
urn:nbn:se:uu:diva-334433 (URN)
Tilgjengelig fra: 2017-11-23 Laget: 2017-11-23 Sist oppdatert: 2017-11-30

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