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1.
Figure 2

Figure 2. Functionally enriched processes identified in MHC-CELFΔ hearts.. From: Gene Expression Analyses Implicate an Alternative Splicing Program in Regulating Contractile Gene Expression and Serum Response Factor Activity in Mice.

The top ten most functionally enriched processes in MHC-CELFΔ versus wild type hearts identified using the Metacore™ pathway analysis database are shown.

Twishasri Dasgupta, et al. PLoS One. 2013;8(2):e56590.
2.
Figure 1

Figure 1. Venn diagram comparisons of affected genes in MHC-CELFΔ hearts.. From: Gene Expression Analyses Implicate an Alternative Splicing Program in Regulating Contractile Gene Expression and Serum Response Factor Activity in Mice.

(A) Respective overlap of affected up-regulated and down-regulated genes between mild and severe MHC-CELFΔ lines. (B) Affected genes in MHC-CELFΔ hearts were compared to affected genes in two cardiac-specific knockout models (SRSF1-ko and SRSF2-ko) lacking one of the SR proteins, SRSF1 or SRSF2 (also known as ASF/SF2 and SC35, respectively).

Twishasri Dasgupta, et al. PLoS One. 2013;8(2):e56590.
3.
Figure 3

Figure 3. The serum response factor (SRF) network is affected in MHC-CELFΔ mice.. From: Gene Expression Analyses Implicate an Alternative Splicing Program in Regulating Contractile Gene Expression and Serum Response Factor Activity in Mice.

The SRF network was identified as the most affected transcription factor network in MHC-CELFΔ mice using Ingenuity Pathway Analysis software. Two up-stream inhibitors of SRF activity, Hopx and Fhl2, are down-regulated (blue circles) in MHC-CELFΔ hearts, while nine down-stream targets of SRF are up-regulated (red circles). Srf levels are unchanged. The saturation level (blue and red circles) reflects the relative fold change in MHC-CELFΔ mice relative to wild type.

Twishasri Dasgupta, et al. PLoS One. 2013;8(2):e56590.
4.
Figure 5

Figure 5. Protein expression of SRF network genes in wild type, mild and severe MHC-CELFΔ lines.. From: Gene Expression Analyses Implicate an Alternative Splicing Program in Regulating Contractile Gene Expression and Serum Response Factor Activity in Mice.

Western blot analysis shows a decrease in expression of the SRF inhibitors HOPX and FHL2 in the mild and severe lines of MHC-CELFΔ mice compared to wild type, accompanied by an increase in the levels of three SRF targets. SRF and GAPDH expression levels are constant. Equivalent loading was further confirmed by Ponceau S staining (data not shown). Representatives of three independent blots with similar results are shown. Irrelevant intervening lanes were excised on the ACTA1 blot.

Twishasri Dasgupta, et al. PLoS One. 2013;8(2):e56590.
5.
Figure 4

Figure 4. Real-time RT-PCR validation of the microarray results for the SRF network genes.. From: Gene Expression Analyses Implicate an Alternative Splicing Program in Regulating Contractile Gene Expression and Serum Response Factor Activity in Mice.

Total RNA was extracted and mRNA levels were assayed using SYBR green-based detection. Reduced levels of upstream inhibitors of SRF and elevated levels of downstream targets of SRF were confirmed in MHC-CELFΔ hearts compared to wild type, while Srf transcript levels remain unchanged. Fold changes shown represent the mean+standard error of the mean of three independent sample sets tested in independent experiments. Similar directional changes or lack thereof were observed for each transcript in all three sample sets. An asterisk indicates the mean is significantly different from that of the wild type (P ≤ 0.05).

Twishasri Dasgupta, et al. PLoS One. 2013;8(2):e56590.
6.
Figure 8

Figure 8. CELF-mediated alternative splicing regulates contractile function via transcriptional and post-transcriptional control mechanisms.. From: Gene Expression Analyses Implicate an Alternative Splicing Program in Regulating Contractile Gene Expression and Serum Response Factor Activity in Mice.

SRF regulates the transcription of genes involved in contractile apparatus structure and function. HOPX and FHL2 bind to SRF and inhibit its ability to activate target gene transcription. We propose a model in which CELF-mediated alternative splicing regulates the SRF transcriptional program via modulation of Hopx and/or Fhl2 levels. Reduced CELF-mediated alternative splicing activity in the hearts of MHC-CELFΔ mice leads to a decrease in Hopx and Fhl2, and a complementary increase in SRF target gene expression. Over-expression of CELF1 in the hearts of MCKCUG-BP1 mice leads to an increase in Fhl2, though not Hopx, and a decrease in some SRF target genes. At this time, it is not clear whether Fhl2 and Hopx transcript levels are regulated directly or indirectly by CELF activity. In addition, CELF proteins regulate the alternative splicing of some cardiac transcripts known to encode proteins involved in contractile function, such as Ank2. Thus, the cardiac CELF-mediated alternative splicing program likely regulates contractile function both by modulating the activity of a key transcription factor that controls the expression levels of contractile genes, and by directing the production of specific variants that affect contractile performance.

Twishasri Dasgupta, et al. PLoS One. 2013;8(2):e56590.
7.
Figure 7

Figure 7. Dysregulation of alternative splicing and SRF inhibitor expression correlates with cardiomyopathy in MHC-CELFΔ males.. From: Gene Expression Analyses Implicate an Alternative Splicing Program in Regulating Contractile Gene Expression and Serum Response Factor Activity in Mice.

(A) Total RNA was harvested from the hearts of adult wild type (WT), unaffected (Un) and affected (Af) MHC-CELFΔ severe line males. The alternative splicing of Mef2A exon 16, Itgb1 exon D, and Ank2 exon 21 were evaluated by RT-PCR. (B) Srf, Hopx, and Fhl2 transcript levels were assessed in unaffected and affected MHC-CELFΔ severe line males by real time RT-PCR. For (A) and (B), mean values+standard error of the mean are shown for three individuals per group. An asterisk indicates the mean is significantly different from that of wild type, whereas a pound sign indicates a significant difference between affected and unaffected males (P ≤ 0.05). Both changes in alternative splicing and transcript levels show a graded response that correlates with the incidence of cardiomyopathy in MHC-CELFΔ males. (C) Total protein was harvested from wild type, unaffected, and affected MHC-CELFΔ severe line males and SRF, HOPX, FHL2, and GAPDH levels were assessed by western blot. HOPX and FHL2 levels are down-regulated to a greater extent in affected than unaffected males, whereas SRF protein levels were similar in unaffected and affected males. Equivalent loading is indicated by constant levels of GAPDH, and was further confirmed by Ponceau S staining (data not shown). Representative blots showing samples from two out of four individuals tested from each group are shown.

Twishasri Dasgupta, et al. PLoS One. 2013;8(2):e56590.
8.
Figure 6

Figure 6. Expression of SRF network genes in wild type and MCKCUG-BP1 hearts.. From: Gene Expression Analyses Implicate an Alternative Splicing Program in Regulating Contractile Gene Expression and Serum Response Factor Activity in Mice.

(A) Total RNA was extracted from adult wild type and MCKCUG-BP1 hearts and mRNA levels were assayed using SYBR green-based detection. A general trend shows an increase in the mRNA levels of Fhl2, the upstream inhibitor of SRF, and a decrease in the levels of some of the downstream targets of SRF in MCKCUG-BP1 hearts compared to wild type, an effect opposite to that seen in MHC-CELFΔ mice. Srf transcripts are also decreased in MCKCUG-BP1 hearts relative to wild type. Fold changes shown represent the mean+standard error of the mean of four independent sample sets tested in independent experiments. An asterisk indicates the mean is significantly different from that of wild type (P ≤ 0.05). (B) Dilutions of total protein from adult MCKCUG-BP1 and wild type hearts were probed on the same blot. Empty or irrelevant lanes between the MCKCUG-BP1 and wild type samples were excised. The level of CELF1 over-expression varied from ∼1.5 fold (CELF1 #1) to ∼2–3 fold (CELF1 #2). An increase in the protein level of the upstream inhibitor of SRF, FHL2, and a decrease in the levels of two downstream SRF targets establish a general pattern opposite to that observed in MHC-CELFΔ hearts. Representatives of three independent sets are shown; similar differences were observed in two out of three MCKCUG-BP1 samples relative to wild type, and correlated with higher levels of CELF1 over-expression. SRF and GAPDH protein levels are unchanged in all samples tested. Equivalent loading was further confirmed on each blot by Ponceau S staining (data not shown).

Twishasri Dasgupta, et al. PLoS One. 2013;8(2):e56590.

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