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The Hmgn Family of Chromatin Binding Proteins Regulate Stem Cell Pluripotency and Neuronal Differentiation

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The Hmgn Family of Chromatin Binding Proteins Regulate Stem Cell Pluripotency and Neuronal Differentiation The Hmgn family of chromatin binding proteins regulate stem cell pluripotency and neuronal differentiation Gokula Mohan, Ohoud Rehbini, Tomoko Iwata, Katherine West Institute of Cancer Sciences, University of Glasgow Hmgn proteins Hmgn proteins are enriched across Hmgn2 is expressed in neural progenitor cells the Oct4 and Nanog gene loci during embryogenesis E11.5 E13.5 E15.5 Bind as dimers to nucleosomes svz Hmgn1 Hmgn2 svz 3 Regulate the deposition of histone 3 3 2.5 modifications 2.5 2.5 2 2 2 1.5 Compete with linker histones 1.5 1.5 1 enrichment 1 1 Interact with several transcription 0.5 0.5 0.5 factors. 0 0 0 svz: ventricular/ -1781 -399 410 2070 3342 -1081 -219 929 1740 2636 Actin Msat c subventricular zone Kato H et al. PNAS 2011;108:12283-12288 Oct4 Nanog H3 dg dg: dentate gyrus (in hippocampus) control cells NLS Nucleosome-binding domain NLS Regulatory domain 2 2 2 very highly conserved Competes with H1 c: cerebellum 1.5 1.5 1.5 100 aa 1 1 1 P14 Inhibits phosphorylation Increases acetylation enrichment 0.5 0.5 0.5 of H3S10 of H3K14 0 0 0 -1781 -399 410 2070 3342 -1081 -219 929 1740 2636 Actin Msat Retinoic acid-induced differentiation of EC P Oct4 Nanog cells down the neuronal lineage. M A A P Reprogramming Differentiation Hmgn1 and Hmgn2 binding profiles in control cells. Enrichment at each PCR set was normalised to Histone H3 N-terminal tail ARTKQTARKSTGGKAPRKQLATKAARKSA... stage stage input and then normalised to average H3 enrichment across all primer sets to control for differences M between chromatin preps. Suspension Adherent Summary: Hmgn1 and Hmgn2 are ubiquitously highly expressed in embryonic tissue, and are progressively +RA -RA downregulated during embryogenesis. Using RNAi, we have ChIP-seq reveals peaks of Hmgn2 and H3 upstream of Nanog -4 -2 0 3 6 9 days shown that Hmgn proteins are essential for the expression of Undifferentiated embryoid bodies Neural differentiation key pluripotency genes, including Oct4 and Nanog, in cells (EBs) embryonal carcinoma cells. ChIP experiments show that levels Oct4 Nanog 1.2 1.2 of H3K4me3 at the promoters of Oct4 and Nanog are Hmgn2 1 1 decreased in Hmgn2 knockdown cells, whereas levels of 0.8 0.8 H3 H3K27me3 are increased. This provides a molecular basis for 0.6 0.6 the observations that an Hmgn2 knockout is embryonic lethal, 0.4 0.4 H3K4me3 0.2 Undiff D3 D6 D9 0.2 and that manipulating the Hmgn content of early Xenopus levels mRNA Relative (public data) embryos causes major developmental defects. 0 0 Nanog Undiff D3 D6 D9 We have also shown that Hmgn proteins are very highly Undiff D3 D6 expressed in neural stem cells, both during embryogenesis and in the adult mouse brain. We studied the effects of Hmgn NF-160 knockdown on the neuronal differentiation of embryonal Hmgn2 carcinoma cells in vitro. The expression levels of various neural stem cell and neuronal genes were altered following H3 Hmgn knockdown, showing that Hmgn proteins are important H3K4me3 DAPI for accurate neuronal differentiation and function. (public data) Oct4 Tcf19 Cchcr1 siRNA knockdown of Hmgn1 or Hmgn2 reduces the expression of pluripotency markers in pluripotent MERGE embryonal carcinoma cells. No No N1a wt -ve siRNA N1 a N1b -ve N2a N2b wt -ve siRNA + N2 a Knockdown of Hmgn2 reduces H3K4me3 on Oct4 and Nanog Hmgn1 • Pluripotency markers Oct4, Sox2 and Nanog are H3K4me3 in control cells repressed during differentiation. Hmgn2 control Hmgn2H3K4me3 kd in Hmgn2 kd cells 25 • Induction of neuronal markers Map2, NF-160, nestin, -Actin 25 10 20 NSE and NMDAR-2 is observed from day 3. 20 8 15 Oct4 Nanog Sox2 15 6 • Induction of glial marker GFAP from day 9. 1.2 2 1.4 10 4 10 1 1.2 enrichment 1.6 5 2 5 1 0.8 Hmgn2 levels increase during 1.2 0.8 0.6 0 0 0 neuronal differentiation of EC cells 0.8 0.6 -1781 -399 410 2070 3342 -1081 -219 929 1740 2636 actin M-sat 0.4 0.4 Oct4 Nanog Undiff D3 D6 D9 0.4 0.2 0.2 Relative mRNA levels levels mRNA Relative 0 0 0 HMGN2 wt -ve N1 N1 N2 N2 N1a + wt -ve N1 N1 N2 N2 N1a + wt -ve N1 N1 N2 N2 N1a + a b a b N2b a b a b N2b a b a b N2b Knockdown of Hmgn2 increases H3K27me3 on Oct4 and Nanog 1.1 3.1 2.3 3.2 Hmgn2/β-Actin siRNA siRNA siRNA β-Actin controlH3K27me3 in control cells PCR array analyses reveal that several markers of early Hmgn2H3K27me3 kd in Hmgn2 kd cells differentiation are upregulated in Hmgn-knockdown EC 0.2 0.3 0.3 cells. The expression of several genes required for ES 0.15 self renewal and pluripotency is also disrupted. 0.2 0.2 0.1 HMGN1 DAPI Merge Upregulated genes in Hmgn knockdown cells enrichment 0.1 0.1 0.05 1 10 100 Fn1 parietal endoderm Cdx2 trophectoderm relative expression 0 0 0 -1081 -219 929 1740 2636 Brachyury mesoderm (log scale) -1781 -399 410 2070 3342 actin Msat Runx2 bone/mesoderm Oct4 Nanog HMGN2 DAPI Merge Noggin mesoderm Hck ES cell Nodal ES cell H3K27me3 on Oct4 and Nanog increases in day 3 neuronal cells Galanin ES cell Lefty2 ES cell wt Kit ES cell H3K27me3 in wt undifferentiated cells Expression of several neuronal genes is significantly Nr5a2 ES cell d3H3K27me3 in wt neuronal cells altered in day 3 immature neuronal cells following Gbx2 ES cell 1 1 Hmgn1 and/or Hmgn2 knockdown. Downregulated genes in Hmgn knockdown cells 1 0.8 0.8 0.8 control Hmgn1 kd Hmgn2 kd 0.01 0.10 relative expression (log scale) 1.00 4 0.6 0.6 Ddx4 germ cell 0.6 3.5 Sycp3 germ cell enrichment 0.4 0.4 0.4 Flt1 endothelial/mesoderm 3 * Ednrb endothelial/mesoderm 0.2 0.2 0.2 Myod muscle/mesoderm 2.5 * Myf5 muscle/mesoderm 0 0 0 2 Desmin muscle/mesoderm -1781 -399 410 2070 3342 -1081 -219 929 1740 2636 actin Msat Oct4 Nanog 1.5 Grb7 ES cell relative expression * * Tcfcp2I1 ES cell 1 * Utf1 ES cell 0.5 0 Rest Map2 Nestin Glyt1a Nse Glyt2 Zfp521 Nmda R2.
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