Extensive NEUROG3 occupancy in the human pancreatic endocrine gene regulatory network.

OBJECTIVE: Mice lacking the bHLH transcription factor (TF) Neurog3 do not form pancreatic islet cells, including insulin-secreting beta cells, the absence of which leads to diabetes. In humans, homozygous mutations of NEUROG3 manifest with neonatal or childhood diabetes. Despite this critical role in islet cell development, the precise function of and downstream genetic programs regulated directly by NEUROG3 remain elusive. Therefore, we mapped genome-wide NEUROG3 occupancy in human induced pluripotent stem cell (hiPSC)-derived endocrine progenitors and determined NEUROG3 dependency of associated genes to uncover direct targets. METHODS: We generated a novel hiPSC line (NEUROG3-HA-P2A-Venus) where NEUROG3 is HA-tagged and fused to a self-cleaving fluorescent VENUS reporter. We used the CUT&RUN technique to map NEUROG3 occupancy and epigenetic marks in pancreatic endocrine progenitors (PEP) that were differentiated from this hiPSC line. We integrated NEUROG3 occupancy data with chromatin status and gene expression in PEPs as well as their NEUROG3-dependence. In addition, we investigated whether NEUROG3 binds type 2 diabetes mellitus (T2DM)-associated variants at the PEP stage. RESULTS: CUT&RUN revealed a total of 863 NEUROG3 binding sites assigned to 1263 unique genes. NEUROG3 occupancy was found at promoters as well as at distant cis-regulatory elements that frequently overlapped within PEP active enhancers. De novo motif analyses defined a NEUROG3 consensus binding motif and suggested potential co-regulation of NEUROG3 target genes by FOXA or RFX transcription factors. We found that 22% of the genes downregulated in NEUROG3-/- PEPs, and 10% of genes enriched in NEUROG3-Venus positive endocrine cells were bound by NEUROG3 and thus likely to be directly regulated. NEUROG3 binds to 138 transcription factor genes, some with important roles in islet cell development or function, such as NEUROD1, PAX4, NKX2-2, SOX4, MLXIPL, LMX1B, RFX3, and NEUROG3 itself, and many others with unknown islet function. Unexpectedly, we uncovered that NEUROG3 targets genes critical for insulin secretion in beta cells (e.g., GCK, ABCC8/KCNJ11, CACNA1A, CHGA, SCG2, SLC30A8, and PCSK1). Thus, analysis of NEUROG3 occupancy suggests that the transient expression of NEUROG3 not only promotes islet destiny in uncommitted pancreatic progenitors, but could also initiate endocrine programs essential for beta cell function. Lastly, we identified eight T2DM risk SNPs within NEUROG3-bound regions. CONCLUSION: Mapping NEUROG3 genome occupancy in PEPs uncovered unexpectedly broad, direct control of the endocrine genes, raising novel hypotheses on how this master regulator controls islet and beta cell differentiation

Prdm5 Regulates Collagen Gene Transcription by Association with RNA Polymerase II in Developing Bone

PRDM family members are transcriptional regulators involved in tissue specific differentiation. PRDM5 has been reported to predominantly repress transcription, but a characterization of its molecular functions in a relevant biological context is lacking. We demonstrate here that Prdm5 is highly expressed in developing bones; and, by genome-wide mapping of Prdm5 occupancy in pre-osteoblastic cells, we uncover a novel and unique role for Prdm5 in targeting all mouse collagen genes as well as several SLRP proteoglycan genes. In particular, we show that Prdm5 controls both Collagen I transcription and fibrillogenesis by binding inside the Col1a1 gene body and maintaining RNA polymerase II occupancy. In vivo, Prdm5 loss results in delayed ossification involving a pronounced impairment in the assembly of fibrillar collagens. Collectively, our results define a novel role for Prdm5 in sustaining the transcriptional program necessary to the proper assembly of osteoblastic extracellular matrix

Genomic and proteomic analyses of Prdm5 reveal interactions with insulator binding proteins in embryonic stem cells.

PRDM proteins belong to the SET domain protein family, which is involved in the regulation of gene expression. Although few PRDM members possess histone methyltransferase activity, the molecular mechanisms by which the other members exert transcriptional regulation remain to be delineated. In this study, we find that Prdm5 is highly expressed in mouse embryonic stem (mES) cells and exploit this cellular system to characterize molecular functions of Prdm5. By combining proteomics and next-generation sequencing technologies, we identify Prdm5 interaction partners and genomic occupancy. We demonstrate that although Prdm5 is dispensable for mES cell maintenance, it directly targets genomic regions involved in early embryonic development and affects the expression of a subset of developmental regulators during cell differentiation. Importantly, Prdm5 interacts with Ctcf, cohesin, and TFIIIC and cooccupies genomic loci. In summary, our data indicate how Prdm5 modulates transcription by interacting with factors involved in genome organization in mouse embryonic stem cells

Prdm5 deregulation impairs osteogenic differentiation <i>in vitro</i>.

<p>A) Upper panel. qRT-PCR of <i>Prdm5</i> levels in MC3T3 cells transduced with lentiviral shRNA constructs against <i>Prdm5</i> (shPrdm5-a and shPrdm5-b) and control construct. Lower panel. Prdm5 western blot from the same experiment. Tubulin is included as loading control. B) Upper panel. Quantification of Alizarin red staining after 21 days of osteogenic differentiation in MC3T3 cells. Data are presented as mean of 3 independent experiments ± SEM. ** = p<0.01 and *** = p<0.005 (t-test). Lower panel. Representative image from osteogenic differentiation experiments. C) Western blot showing overexpression of human PRDM5 (marked with star) in MC3T3 cells (filled circle = endogenous Prdm5). GFP overexpression is used as a negative control and Vinculin western blot for equal protein loading. D) Quantification of Alizarin red staining from osteogenic differentiation experiments of MC3T3 cells overexpressing GFP or PRDM5. A representative experiment is shown and data are presented as average ± standard deviation. E) qRT-PCR analysis of WT and <i>Prdm5^LacZ/LacZ</i> calvaria osteoblasts for osteogenic markers as indicated. Expression values were normalized to a panel of housekeeping genes (<i>Rps18, Ubc, Actb, Rpl0</i>) and indexed to the average expression value of wild type clones. * = p<0.05 and *** = p<0.001, by unpaired T-test, +/+ (n = 14), LacZ/LacZ (n = 19).</p

Analyses of Prdm5 chromatin interactions.

<p>A) Diagram illustrating the overall distribution of Prdm5 binding sites categorized according to the distance from the nearest TSS (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002711#pgen.1002711.s010" target="_blank">Text S1</a>). B) The mean distribution of tags across gene bodies for Prdm5 ChIP-seq (Prdm5-ab1 in blue, Prdm5-ab2 in red and IgG in black). Vertical dashed line at x = 0 represents the TSS. Positions after the TSS are represented as % of the length of the gene. C) Upper panel. Slogos plot produced using the motifs detected by the Weeder program from Prdm5 “shrunk” peaks (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002711#pgen.1002711.s010" target="_blank">Text S1</a>). Lower panel. DNA pulldown assay from nuclear extract of 293 cells overexpressing HA-PRDM5 using biotinylated oligos representing the <i>Col1a1</i> exon 33 (WT) and a mutated control sequence (G-A/T Mut). D) Histogram showing the percentage of H3K9me3 (left panel), H3K4me3 (middle panel) and RNA Polymerase II (right panel) positivity for “Random sampling” (mean value of 100 iterations for 1446 random genes sets) or for Prdm5 target genes. (E) Q-Q plot comparing the quantile distribution of Prdm5 target genes' expression (on Y axis) and all genes (on X axis). Red line is reference line representing equal quantile distribution.</p

Prdm5 regulates <i>Decorin</i> through a distal enhancer.

<p>A) ChIP-qPCR validation of Prdm5 peaks on selected ECM genes or negative regions from an independent sample immunoprecipitated with IgG, Prdm5-Ab1 (in black and blue respectively, plotted on left Y axis) or Prdm5-Ab2 (in red, plotted on right Y axis). Orange horizontal line represents the highest “noise” value obtained by ChIP-qPCR on a set of negative regions. B) qRT-PCR analysis of <i>Decorin</i> transcript (<i>Dcn</i>) levels upon Prdm5 knockdown as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002711#pgen-1002711-g004" target="_blank">Figure 4E</a>. Results are presented as average of four independent experiments +/− SD; * = p<0.05. (C) qPCR analysis of WT and <i>Prdm5^LacZ/LacZ</i> calvarial osteoblasts for <i>Decorin</i>. Expression values were normalized to the control WT samples. *  = p<0.05; T-test, (+/+ n = 14, LacZ/LacZ n = 19 clones). D) Upper panels. Western blot analysis of Decorin levels upon Prdm5 knockdown in cell layers; Tubulin is used as loading control. Lower panels. Western blot analyses of purified proteoglycans from cell culture media from knockdown cells. Tubulin is used as purity control and Fibronectin for equal protein loading. E) ChIP-qPCR with indicated antibodies for Prdm5 binding site upstream of Dcn gene. Meg3 TSS region is used as negative control.</p

Prdm5 targets all collagen genes and regulates type I collagen expression.

<p>A) ChIP-qPCR validation of Prdm5 peaks in collagen genes or negative control regions from an independent chromatin preparation immunoprecipitated with IgG, Prdm5-Ab1 (in black and blue respectively, plotted on left Y axis) or Prdm5-Ab2 (in red, plotted on right Y axis). Orange horizontal line represents the highest “noise” value obtained by ChIP-qPCR on a set of negative regions. B) Biological processes enrichment from gene ontology annotation of Prdm5 target regions. C) Distribution of the “peak centre” position for collagen genes targeted by Prdm5 or random sampling of Prdm5 target genes according to genetic feature. D) Correlation between Prdm5-Ab1 coverage inside the gene body of all mouse collagen genes (X-axis) and Pol II coverage in the same regions (Y-axis) normalized by base pairs. E) qRT-PCR for <i>Prdm5</i>, <i>Col1a1</i> and <i>Col1a2</i> in MC3T3 cells treated for 72 hours with siRNA oligos against <i>Prdm5</i> (siPrdm5-1 and -2) or controls. Results are presented as average of four independent experiments ± SD; * = p<0.05, ** = p<0.01. F) Upper panel. ChIP-qPCR for RNA Pol II in WT and mutant (blue and red respectively) calvarial osteoblasts along the <i>Col1a1</i> gene. IgG control is represented by black and green lines respectively. X-axis = distance (in bp) from <i>Col1a1</i> TSS, * = p<0.05 (unpaired t-test). Lower panel. Genome browser snapshot of the corresponding <i>Col1a1</i> genomic region displaying MC3T3 tracks for: qPCR amplicons, IgG, RNA Pol II and Prdm5-Ab1 coverage. G) Western blot from co-immunoprecipitation experiment of HA-PRDM5 in HEK293 cells; endogenous interacting proteins or IgG are indicated.</p

Prdm5 is expressed in osteoblast regions of developing bones.

<p>A) Scheme for the generation of the <i>Prdm5^LacZ/LacZ</i> mouse strain. B–E) X-gal stainings of <i>Prdm5^LacZ/LacZ</i> embryos at E10.5 (B), E12.5 (C) E14.5 (D) and E16.5 (E). F) E16.5 embryo image detail. LacZ reporter expression in the perichondrium and growth plate of femur and ribs is marked by arrows. G) X-gal staining of tibiae section from E16.5 <i>Prdm5</i> mutant embryo. Juxtaposition of three pictures (separated by white lines) to represent the whole length of a tibia. Indicated are different compartments: PC = proliferative chondrocytes, HC = hypertrophic chondrocytes, OB = osteoblasts. Periosteum is marked by asterisks. H) Whole mount X-gal staining of <i>Prdm5^LacZ/LacZ</i> newborn skull at P0. Pronounced staining in sutures is indicated with an arrow. Bars = 1 mm, except for (G) where bar = 200 µm.</p