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Study of the regulation of ACVR1 expression

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(1)

Renata Bocciardi, Serena Cappato, Francesca Giacopelli, Marzia Mura Prof. Roberto Ravazzolo

S.C. Genetica Molecolare e Citogenetica Istituto G. Gaslini – Genova

Dipartimento di Scienze “Pediatriche G. de Toni”

Università degli Studi di Genova

Study of the regulation of ACVR1 expression

(2)

 5’UTR sequences

 Promoter region

 3’UTR region

Study of the regulation of ACVR1 expression

Characterization of the structure and composition of the ACVR1 transcripts.

Definition and characterization of sequences playing a role in the regulation of ACVR1 expression.

(3)

Study of the regulation of ACVR1 expression

Characterization of the structure and composition of the ACVR1 transcripts.

Definition and characterization of genomic regions playing a role in the regulation of ACVR1 expression, such as gene promoter/s and/or other sequences with regulatory activity.

Promoter region 5’ UTR Protein coding region 3’ UTR

ATG STOP

mRNA 1 mRNA 2

transcription

translation

protein Transcriptional regulation

Promoter

Enhancer/silencer

Post-transcriptional regulation

Splicing

mRNA stability/availability

Small RNA

Effect on translation

(4)

Study of the regulation of ACVR1 expression

Characterization of the structure and composition of the ACVR1 transcripts.

Definition and characterization of sequences playing a role in the regulation of ACVR1 expression.



5’UTR sequences

Promoter region 3’UTR region

(5)

 Our bioinformatic analysis suggests the putative presence of different transcription start sites (green and blue stars) and of different transcripts showing the alternative combination of several 5’UTR exons (white rectangles) with common protein coding sequences (violet rectangles, only the first two out 9 protein coding exons are indicated).

 The 5’UTR region of the ACVR1 gene that undergoes alternative splicing spans around 57 kb of genomic DNA. Untranslated exons are flanked by canonical splicing sites and show a different degree of conservation.

ATG AL700573?

Under analysis Under analysis ATG

ATG L02911

ENST00000412025

ATG NM_001105 ?

ATG ENST00000409283

ENST00000487456 ?

NM_001111067

DB270850 DA995330 DA436676

?

ATG ATG ATG ATG

Characterization of the structure and composition of the ACVR1 transcripts.

How many are ACVR1 transcripts?

(6)

ACVR1 is widely expressed.

The most common cDNA isoform is that defined by the NM_001111067 RefSeq.

 We have identified a new NM_001111067 isoform carrying an insertion at the 3’ end of the second UTR exon (NM_001111067plus).

fBc fL fLi fK fH fS fT fSk -

cDNA from fetal tissues cDNA from adult tissues

B S HH K L PBL -

cDNA from commonly used cell lines

ATG

NM_001111067 NM_001111067 plus*

ATG

Characterization of the structure, composition and expression profile of the ACVR1 transcripts (I)

(7)

 The insertion is due to an alternative splicing that leads to the incorporation of 116 bp of the 5’end of the adjacent intron in the mRNA.

 Although quantitatively less represented, also this isoform is widely expressed as assessed by RT-PCR on different cDNA with a forward oligonucleotide specific for the insertion (*) combined to the reverse mapping in the coding sequence (*).

 The inserted sequence is highly conserved among species and is even more conserved than the adjacent exon.

Characterization of the structure, composition and expression profile of the ACVR1 transcripts (II)

cDNA from adult tissues

B S H K L PBLHeLa -

* *

NM_001111067 NM_001111067 plus*

ATG ATG

(8)

DA995330 DA436676

cDNA from fetal tissues cDNA from adult tissues

fB fL fLi fK fH fS fT fSk

fB fL fLi fK fH fS fT fSk

B S H K L PBL

Saos-2 BMSC

B S H K L PBL

BMSC -

 We could obtain specific RT-PCR products also for isoforms corresponding to DA436676 and DA995330 ESTs.

 We found that DA995330 EST is also subjected to alternative splicing

NM_001111067 ATG

NM_001111067 ATG

plus*

DA436676 ATG

DA995330* ATG

ATG

LTR LTR

ATG

DA995330**

DA995330***

Characterization of the structure, composition and expression profile of the ACVR1 transcripts (III)

(9)

ACVR1 gene shows several transcripts due to alternative splicing combining different 5’UTR sequences to the common protein coding region.

These alternative isoforms are apparently less expressed compared to the main transcript.

What is the role of these different 5’UTR sequences?

5’UTR regions can be involved in regulating gene expression at post

transcriptional level by different mechanisms, for example, by forcing mRNA in a stable stem-loop secondary structure that may prevent an efficient scanning of the ribosome along the 5’UTR in search for the primary ATG start codon.

5’UTRs may affect the mRNA stability.

(10)

Full-lenght ACVR1 cDNA carrying the different 5’UTR sequences were subcloned in the pCMV3-Tag8 expression vector in frame with a 3xFLAg epitope.

Constructs were transiently transfected in U2OS cells together with a pRL expression vector for Renilla luciferase as transfection control. After 48 h cells were lysed and analysed by western blot.

ACVR1 protein expression was normalized to GAPDH levels (as loading control) and to Renilla firefly activity (as transfection efficiency control).

NM_001111067 ATG

ATG

NM_001111067 plus*

DA436676 ATG

DA995330 ATG

DA995330 NM_001111067

NM_001111067plus

DA436676

ACVR1 cDNA 3xFLAG

3xFLAG ACVR1 cDNA

3xFLAG ACVR1 cDNA

3xFLAG ACVR1 cDNA

3xFLAG

Without 5’UTR

(11)

No UTR 067

067plus DA995

DA436 ev

α αα

α-FLAG

α α α

α-GAPDH

DA995330 NM_001111067

NM_001111067plus

DA436676

ACVR1 cDNA 3xFLAG

3xFLAG ACVR1 cDNA

3xFLAG ACVR1 cDNA

3xFLAG ACVR1 cDNA

3xFLAG

Without 5’UTR 0 20 40 60 80 100 120

O67 O67plus DA995 DA436

Densitometric analysis of WB

ACVR1 encoded by cDNAs carrying the different 5’UTR

sequences are differently expressed

(12)

We have characterized the structure and composition of ACVR1 transcripts that show a complex organization at the 5’UTR end of the gene.

We have identified a transcription start site common to all isoforms experimentally verified

The role of these sequences has to be clarified, they may intervene in

negative modulation of ACVR1 expression at post transcriptional level (upon specific stimuli?) as suggested by the different expression of the alternative constructs in transfection experiments.

Identification of this transcription start site gave us the opportunity to characterize the ACVR1 promoter region.

(13)

Study of the regulation of ACVR1 expression

Characterization of the structure and composition of the ACVR1 transcripts.

Definition and characterization of sequences playing a role in the regulation of ACVR1 expression.

 5’UTR sequences



Promoter region

3’UTR region

Generation of a cellular model suitable for the high through-put

screening of small molecules with potential pharmacological effect on

the BMP/ACVR1 mediated pathway.

(14)

Identification of the ACVR1 promoter

•The ACVR1 promoter has not been identified yet.

•To this aim we have selected and isolated a genomic region of around 2.8 kb mapping upstream the verified transcription start site.

•This region has 55% of GC nucleotides clustered in long stretches and predominantly mapping in the proximal 1 kb segment.

•No TATA –boxes are present.

•Several binding sites for different transcription factors are predicted by the in silico analysis (Sp1, SMAD, KLF...).

The region appeared to be well conserved in mouse.

(15)

Vista Genome browser tracks comparing human versus mouse genome of the selected 2.8 kb upstream the ACVR1 transcription start site and harbouring different well

conserved sequences (red peaks)

+1 identified transcription start site

(16)

Functional study of ACVR1 promoter

-2893 +32

Luc

ACVR1 gene pPrACVR1-2.8

Subcloning of the 2.8 kb fragment in a luciferase expression vector (pGL3- basic vector).

Transfection in different cell lines and evaluation of the Luciferase activity.

(17)

U2OS

0 20 40 60 80 100 120

Luciferase Activity 1pGL3 Promoter Vector pPrACVR1-2.8

C2C12

0 10 20 30 40 50 60 70

Luciferase Activity 1pGL3 Promoter Vector pPrACVR1-2.8

The selected fragment showed a strong promoter activity in different cell lines.

0 20 40 60 80 100 120

Luciferase activity 1pGL3 Promoter Vector pPrACVR1-2.8

HeLa

We decided to continue the analysis by generating different deletion constructs to be used in further transfection experiments.

(18)

pPrACVR1-1.4 pPrACVR1-1.2 pPrACVR1-0.7 pPrACVR1-0.3 pPrACVR1-2.8

Prom Vec

0 50 100 150 200 250 300 350 400 450

Luciferase activity

pPrACVR1-1.2 p

+32

-1462 -1158

-762 -308

-2893

Functional analysis of ACVR1 promoter deletion constructs (I)

(19)

0 20 40 60 80 100 120 140 160 180

Luciferase activity

pPrACVR1-1.2 p

+32

pPrACVR1-0.072 pPrACVR1-0.16 pPrACVR1-0.3 pPrACVR1-0.4 pPrACVR1-0.6 pPrACVR1-0.7 Prom Vec

Functional analysis of ACVR1 promoter deletion constructs (II)

(20)

pPrACVR1-1.2 p

+32

pPrACVR1-0.072 pPrACVR1-0.16 pPrACVR1-0.3 Prom Vec

0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6

Luciferase activity

Functional analysis of ACVR1 promoter deletion constructs (III)

(21)

We have characterized a 2.8 kb genomic region with strong promoter activity upstream the identified transcription start site.

Sequence from -308 to +1 still has a detectable promoter activity ranging from 20% to 60%, depending on the cell lines used, of the activity of a strong viral promoter (SV40) used as control

Effect of the cell density on ACVR1 promoter

0 5 10 15 20 25 30 35

pPrACVR1-2.8 pPrACVR1-1.4

Luciferase activity

Low cell density High cell density

C2C12

0 5 10 15 20 25

pPrACVR1-2.8 pPrACVR1-1.4

Luciferase activity

Low cell density High cell density

U2OS

The ACVR1 promoter activity controlled by the two longest fragments seems to be sensitive to cell culture conditions such as cell densitiy

(22)

Rosi and FOP

(23)

Rosiglitazone

Rosiglitazone (Rosi) belongs to the class of Thiazolidinediones (TZDs) and is an orally active hypoglycemic agent for the treatment of non-insulin-dependent diabetes mellitus or Type 2 Diabetes (T2D).

Rosi, as the other TZDs, is a known agonist of the Peroxisome Proliferator Activated Receptor γ (PPARγ) .

PPARγ is a ligand dependent transcription factor that belongs to the family of nuclear hormone receptors.

It is involved in several different physiological and pathological processes such as differentiation, inflammation, aging, obesity, infertility and cancer.

In particular, it is a key molecule for adipogenesis, and is important for glucose homeostasis and control of inflammation.

(24)

Is there a biological basis for treatment of Fibrodysplasia Ossificans Progressiva (FOP) with Rosiglitazone?

PPARγ shows two relevant features

(I) PPARγ is a potent anti-inflammatory molecule.

(II) PPARγ affects bone metabolism, inhibits osteogenesis by switching cell differentiation towards adipogenesis.

Rosi and ACVR1

1) Does Rosi affect ACVR1 gene espression?

2) Does it affect the ACVR1 mediated BMP pathway?

(25)

Set up of cell treatment with Rosi

•Transient transfection of an expression construct for PPARg (pSG5-PPARg) together with a Luciferase expression vector driven by a PPARγ responsive element (PPRE-Luc).

•Treatment of cells with different doses of Rosi for 24 h.

0 20 40 60 80 100 120 140 160 180

Luciferase activity

DMSO 1 µµµµM 5 µµµµM 10 µµµMµ Rosi

Rosi and ACVR1

Transient transfection of PPARg (pSG5-PPARg) expression vector with the Luciferase reporter construct carrying the different ACVR1 promoter fragments.

Treatment of transfected cells with 1 µM Rosi (+) or DMSO (-) for 24h.

Detection of the Luciferase activity.

(26)

0 10 20 30 40 50 60 70 80

Luciferase activity

- + - + - + - + - +

pPPARγγγγ/PPRE-luc

pPrACVR1-2.8

pPrACVR1-0.7 pPrACVR1-1.4

pPrACVR1-1.2

Effect of Rosi treatment on ACVR1 promoter:

fragment from – 2800 bp to -700 bp

(27)

0 5 10 15 20 25 30

Luciferase activity

- +

pPrACVR1-0.7

- +

pPrACVR1-0.6

- +

pPrACVR1-0.3

- +

pPrACVR1-0.16

- +

pPrACVR1-0.072

Effect of Rosi treatment on ACVR1 promoter:

fragment from – 700 bp to -72 bp

(28)

0 10 20 30 40 50 60 70 80 90 100

% of activity reduction

pPrACVR1-2.8

pPrACVR1-1.4

pPrACVR1-1.2

pPrACVR1-0.7

pPrACVR1-0.6

pPrACVR1-0.3

pPrACVR1-0.16

pPrACVR1-0.072

In accordance, cells transfected with PPARγγγγ expression vector and treated with Rosi showed a reduction in the endogenous expression of ACVR1 gene

(as assessed by qRT-PCR experiments).

Rosi treatment induces a reduction in ACVR1 promoter activity.

Activity of all deletion fragments is inhibited of around 40% upon Rosi treatment. Inhibitory effect is maintained up to a region as short as 72 bp upstream of the transcription start site.

(% of reduction is calculated from different independent experiments, respect to the activity obtained for each fragment in untreated cells considered as 100%).

(29)

PPARγ transcriptional activity, either positive (transactivating) or negative

(transrepressing), is modulated both by the action of a ligand/agonist and by post- translational modifications.

Direct transcriptional regulation of target genes is usually dependent on the obligate dimerization with RXR factors and DNA binding at specific sites.

However, in silico analysis of the ACVR1 promoter sequence has revealed the

presence of a unique PPARγ/RXR binding site probably not involved in the ACVR1 responsiveness to Rosi.

Therefore, we postulated that the transcriptional regulation operated by Rosi/PPARγ might be mediated by interaction with others transactivating factor(s) recruited to the ACVR1 promoter and/or cis-regulatory element(s).

(30)

+32

Sp1 binding sites (1/1, core/matrix score) Sp1 binding sites (1/>0.9, core/matrix score) PPARg/RXR binding site

 ACVR1 promoter sequence has 55% of GC nucleotides clustered in long stretches and “GC- boxes”.

 In silico analysis predicted the presence of several binding sites for Sp1 transcription factor.

(31)

 Sp1 is a well known, ubiquitous nuclear transcription factor involved in multiple biological processes (control of cell growth, differentiation, apoptosis etc.).

 Sp1 binds to GC-rich sequences and can regulate the transcription of housekeeping and of TATA-less promoter genes.

 Recent studies have shown that DNA-binding and transcription activity of Sp1 change in response to post-translational modifications induced by

extracellular stimuli through a variety of signal transduction pathways.

 Moreover, several studies indicate that Sp1 can be involved in the

transcriptional repression of target genes (resistin, follistatin, α1(I) collagen...) induced by TZDs.

 Indeed, TZD-activated PPARγ can be recruited to the promoter region of target genes through the interaction with Sp1 and thus can exert its

repression activity also on those promoters lacking specific PPARγ/RXR binding sites.

(32)

0 100 200 300 400 500 600 700 800

Luciferase activity

+32

pPrACVR1-2.8 pPrACVR1-1.4

pPrACVR1-1.2 pPrACVR1-0.7

pPrACVR1-0.16 pPrACVR1-0.072

Empty vector Sp1

DN1

CMVSP1

Does Sp1 regulate the transcription of ACVR1 gene?

Co-tranfection of U2OS cells with reporter contructs for the ACVR1 promoter fragments with expression vector for wild-type Sp1 transcription factor (SP1) or an Sp1 dominant negative form with impaired transactivating function (DN1).

 With longest constructs Sp1 overexpression shows small effect on ACVR1 transcription, while the effect of the DN Sp1 is well detectable.

 With shorter constructs both effects are more evident suggesting that Sp1 binding elements predicted in the proximal promoter sequence play a relevant role in ACVR1 transcriptional regulation.

(33)

Conclusions

We have characterized the structure and composition of ACVR1 transcripts that show a complex organization at the 5’UTR end of the gene.

These sequences may intervene in modulation of ACVR1 expression at post transcriptional level and we are currently investigating in this direction.

We have identified a transcription start site common to all isoforms experimentally verified

We have characterized a 2.8 kb genomic region with strong promoter activity upstream the identified transcription start site. Deletion constructs containing different fragments of the region show a strong activity in the proximal 700 bp. We have also defined a basal promoter region of 72 bp upstream the tss.

We have demonstrated that ACVR1 promoter activity is inhibited by Rosi treatment. Our preliminary results suggest that Sp1 expression can be involved in ACVR1 promoter regulation.

Data from the literature indicate that TZD-activated PPARγγγγ can mediate gene repression through cross-coupling with SP1 and...we are currently

investigation also in this direction...

(34)

Acknowledgements

We thank FOP patients and their families, in particular we are grateful to the "FOP Italia Association" for their enthusiastic

collaboration and the financial support to our research.

This work was supported by Grant 2007RLLET8 of the Italian Ministry of University and by the Strategic grant RFPS-2007-4-631972

of the Italian Ministry of Health.

(35)

Effect of the cell density on ACVR1 promoter

0 5 10 15 20 25 30 35

pPrACVR1-2.8 pPrACVR1-1.4

Luciferase activity

Low cell density High cell density

C2C12

0 5 10 15 20 25

pPrACVR1-2.8 pPrACVR1-1.4

Luciferase activity

Low cell density High cell density

U2OS

ACVR1 promoter activity is sensitive to cell culture conditions and to cell densitiy

(36)

0 5 10 15 20 25 30 35

Luciferase activity

- +

pPrACVR1-2.8

- +

pPrACVR1-1.4

- +

pPrACVR1-1.2

- +

pPrACVR1-0.7

(37)

+32

-1462 -1158

-762

-308

-2893

-581

-131 -72

pPrACVR1-2.8 pPrACVR1-1.4

pPrACVR1-1.2 pPrACVR1-0.7

pPrACVR1-0.6 pPrACVR1-0.3

pPrACVR1-0.16 pPrACVR1-0.72

PPARg RXR

SP1

(38)

Does Sp1 regulate the transcription of ACVR1 gene?

pPrACVR1-2.8

pPrACVR1-1.4

pPrACVR1-1.2

pPrACVR1-0.7

pPrACVR1-0.16

pPrACVR1-0.072

0 100 200 300 400 500 600 700 800

Luciferase activity

Co-tranfection of U2OS cells with reporter contructs for the ACVR1 promoter fragments with expression vector for wild-type Sp1 transcription factor (SP1) or an Sp1 dominant negative form with impaired transactivating function (DN1).

Our preliminary results suggest that Sp1 can regulate ACVR1 promoter transcription

+32

Empty vector Sp1

DN1

CMV SP1

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