Garza Manero, Sylvia

Gurden, Ross

Iwata, Tomoko

Mohan, Gokula

Rehbini, Ohoud

Sindi, Abdulmajeed

West, Katherine

English

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Knockdown High Mobility Nucleosomal Binding Proteins 2 (HMGN2) Alter the Histone Modification H4K4me3 and H3K27me3 and Regulates Stem Cells Pluripotency

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mHmgn1 and Hmgn2 expression in the mouse brainVZ: ventricular zone; SVZ; subventricular zone; DG: dentate gyrus ( in the hippocampus)Ohoud Rehbini, Gokula Mohan, Abdulmajeed Sindi,  Sylvia Garza Manero, Ross Gurden, Tomoko Iwata and Katherine L. West.Institute of Cancer Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, UKKnockdown High mobility nucleosomal binding proteins 2 (HMGN2) alter the histone modification H4K4me3 and H3K27me3 and regulates stem cells pluripotency. HMGN2 HMGN1 P14 E11.5 DG VZ/SVZ VZ/SVZ VZ/SVZ DG VZ/SVZ 0 0.5 1 1.5 undif 1DAI 2DAI 3DAI 4DAI 5DAI 6DAI 8DAI 10DAI 16DAI HMGN1 0 0.5 1 1.5 2 2.5 undif 1DAI 2DAI 3DAI 4DAI 5DAI 6DAI 8DAI 10DAI 16DAI HMGN2 Western blot data from two different experiments indicate a reduction inthe expression of Hmgn1 and Hmgn2 after neural induction. Both proteinsare found at high levels in undifferentiated ECCs, and are nearlyundetectable in terminal stages of differentiation. The Hmgn2 mRNAlevels are consistent with the western blot results, decreasing during thedifferentiation process. However, Hmgn1 mRNA seems to be fluctuatingthrough the different time points analyzed.0.00%20.00%40.00%60.00%80.00%100.00%120.00%no puro 2d puro 1.5ug5d puro 1.5ug10d puro 2ug/ml 15d puro 2ug/ml 20d puro 2.5ug/ml25d puro 2.5ugthe green cells percentage non-silencing-20Hmgn2-968-20Hmgn2-318-20Hmgn2-514-20Merge       green florescent     bright field  Non-silencing   381 (Hmgn2)Knockdown of Hmgn2 using a lentiviralsystem (pGIPZ) in P19 cell linesHMGN2Data from the Allen Brain Atlas indicates that Hmgn1and Hmgn2 mRNAs are highly expressed in activeneurogenic regions of the developing and adult mousebrain, including the subventricular zone and thedentate gyrus of the hippocampus.P19 cells were transduced with lentiviruses expressing three different shRNAmir triggers against Hmgn2 or a control. Cells were selected with puromycin, and FACS analysis shows the percentage of positive transduced cells. Transduced cells express GFPFluorescent microscopy on day 20 for P19 cells transduced with the 381 shRNAmiR trigger against Hmgn2.  Gene expression data from transduced cells, 20 days after transduction. The data show a knockdown in HMGN2 levels, no change in HMGN3, and an increase in HMGN1. Consistent with the data from siRNA transfections, HMGN2knockdown was associated with reductions in Nanog and Oct4expression. However, Sox2 expression was increased in thelentiviral experiment rather than decreased.NanogOct4Actin 0 DAI3 DAI10 DAI16DAI8 DAI6 DAIβ-III Tubulin0DAI3 DAI4 DAI6 DAI10 DAI14 DAI8 DAIActin NF-160MAP2Adherent monolayer culture efficiently drives production of  NPCs and neurons from P19 ECssiRNA knockdown of Hmgn1 or Hmgn2reduces the expression of pluripotency markers in pluripotent P19 EC cells.PCR array analyses reveal that several markers of early differentiation are upregulated in Hmgn-knockdown  EC cells. The expression of several genes required for ES self renewal and pluripotency is also disrupted.00.511.522.53non-silencing 318hmgn2Hmgn1Hmgn3bsox2nanogOCT4Pluripotency gene expression is altered in Hmgn2 knockdown cellsAcknowledgments We would like to thank Dr. Adam West, and all his lab members, for all advice and scientific discussionsFunding for this project  was provided by the Saudi Arabian Ministry of Education (Albah University). the Ministry of Education in Saudi Arabia (King Abdullah scholarship programme), the Malaysia government, and CONACyT.IntroductionHigh mobility group nucleosome binding proteins (HMGN) are ubiquitous non-histone chromosomal proteins. They play important roles in regulating chromatin architecture and transcription. There are five family members: Hmgn1, Hmgn2, Hmgn3a/b, Hmgn4 and Hmgn5. Hmgn1 and Hmgn2 expression is reduced during neuronal differentiation in vitro HMGN proteins bind to nucleosomes with their highly conserved nucleosome binding domain (NBD). The C-terminal regulatory domain (RD) modulates specific histone modifications and disrupts the binding of linker histones (H1) to chromatin. Several studies have shown that HMGN1 and HMGN2 are highly expressed in tissue-specific progenitor and transit-amplifying cells, and are down-regulated in terminally differentiated cells. Functional studies have shown that they play important roles in several cellular differentiation pathways, including erythropoiesis, myogenesis, osteoblast differentiation.Consistent with the expression pattern data, a previous study1 has shown that Hmgn1 is important for regulating the differentiation of neural stem cells, both in vitro and in vivo. Our immediate aim is to develop an in vitro model system with which to investigate the role of HMGN proteins and epigenetic processes during neuronal differentiation. Here, using an adherent culture system2, we demonstrate that the expression of HMGN1 and HMGN2 proteins decreases during the terminal differentiation of neurons from P19 embryonic carcinoma cells.1 Nagao et al (2014) Stem cells 32(11):2983-972 Nakayama et al (2014) Journal of Neuroscience Methods. 227; 100–106.Hmgn1-ActinHmgn2wt     -ve      No siRNA      N1a N1bN1a +N2awt -veNo siRNA   -ve     N2a N2b00.40.81.21.62wt -ve N1a N1b N2a N2b N1a + N2bRelative mRNA levels 00.20.40.60.811.200.20.40.60.811.21.4wt -ve N1a N1b N2a N2b N1a + N2bwt -ve N1a N1b N2a N2b N1a + N2bOct4 Nanog Sox2siRNA siRNA siRNAIn order to verify the results from the transient siRNA experiments, HMGN2 was knocked down using shRNAmiRexpressed from lentiviral vectors.HMGN2 KD cells show reductions in Oct4 and Nanog expressionmRNA expressionConsistent with the data from qRT-PCR and western blotting of whole cell extracts, reductions in Hmgn2, Oct4 and Nanog were observed in chromatin from cells transduced with the 318 trigger. Notably, Sox2 and c-Myc were also downregulated, whereas Klf4 was upregulated. A slower migrating form of TBP was observed in the 318 knockdown cellsIn order to study the levels of histone modifications and DNA bound factors, chromatin was prepared from transduced cells. After reversing the crosslinks, samples were run on SDS-PAGE and analysed by western blotting.We have presented data from two independent approaches indicating that Hmgn2 plays a role in regulating key pluripotency genes such as Oct4, Nanog and c-Myc in mouse embryonal carcinoma cells.Moreover we found that the loss of Hmgn2  compensate with the Hmgn1 at the same location in mRNA , western blot and ChIP-QPCR analysis. Enhancer is marked by Histone post translational modification (H3K4me3). this indicate that Hmgn2 may affect the enhancer region at the active genes. Work is currently ongoing to investigate whether Hmgn2 regulates expression of pluripotency genes in mouse ES cells as well as in EC cells. Hmgn2 binds at the enhancer of active genes   Actin Hmgn2Hmgn10 3 6 8 10 16 daysP19 cells in advanced DMEM/ F12 containing serumNeural InductionDMEM/F12 (no serum) with: 500nM Retinoic acidN2FGF8DAPTCell Culture Vessels coated with 5 mg/ml lamininNBA media with B27 and 8 mMAraCNBA media with only B27Day 0 Day 4Day  2 Day 6Axogenesis Synaptogenesis Synapse  maturationUndiff. P19Western blotting of whole cell extracts confirms Hmgn2 knockdown and loss of Nanog and Oct4 expression. 00.511.522.5rest MAP2 nestin glyt1a NSE Glyt2 zfp521Relative mRNA expression-vesiN1siN2Hmgn1 and Hmgn2 regulate gene expression in day 3 neuronal cells* **** P < 0.05Gene expression data from Hmgn1 and Hmgn2 knockdown after neuronalindication on day 3. these genes are not expressed in undifferentiated P19EC cells. interestingly, GlyT2 expression is highest on day 3 of neuraldifferentiation. HMGN2 knockdown during neural differentiation reducedGlyT2 expression by 75%, whereas the HMGN1 knockdown reducedGlyT2 expression by about 40% compared to wild type cells. In contrast,there was a trend towards increased expression of GlyT1a in the HMGN1and HMGN2 single knockdowns. The expression of Zfp521 is down-regulated upon HMGN1 and HMGN2 knockdown .1   2   3          Hmgn2Hmgn1NanogOct4sox2Actin1. WT P192. NON-SILENCING3. shRNA 381 1     2       3     4       5      61. WT P192. Mouse ES cells3. 381 (Hmgn2 trigger)4. Non-silencing trigger1  2   3  4   5      6DNA gel1 2 3 4500 bpChIP-qPCR analysis in Hmgn2knockdown cells 1    2   3 4c-MycKlf4Sox2Oct4TBPHmgn1Hmgn2Nanog012345678OCT1. OCT2. OCT3. OCT4. OCT5. ACTIN MSATHmgn2 relative enrichment oct4 promoter sites NON-SILENCINGshHMGN2-3810123456N1 N2 N3 N4 N5 ACTIN MSATHmgn2 relative enrichment Nanog promoter sites Non-silencingshHMGN2-3810123456789OCT1. OCT2. OCT3. OCT4. OCT5. ACTIN MSATHmgn1 relative enrichment oct4 promoter sites non-silencingshHMGN2-3810123456789N1 N2 N3 N4 N5 ACTIN MSATHmgn1 relative enrichment Nanog prmoters Non-silencingshHMGN2-381QPCR analysis of Oct4 and Nanog around transcription stating sites(TSS) after Hmgn1 and Hmgn2 ChIP. Global loss of Hmgn2 andenrichment of Hmgn1 around Oct4 and Nanog TSS in Hmgn2knockdown P19 EC cells.00.511.522.53OCT1. OCT2. OCT3. OCT4. OCT5. ACTIN MSATH3K27me3 relative enrichment oct4 promoter sites non-silencingshHMGN2-38100.511.522.53N1 N2 N3 N4 N5 ACTIN MSATH3K27me3 relative enrichment Nanog promoter sites Non-silencingshHMGN2-38100.20.40.60.811.21.41.6OCT1. OCT2. OCT3. OCT4. OCT5. ACTIN MSATH3K4me3 relative enrichment Oct4 promoter sites non-silencingshHMGN2-38100.511.522.5N1 N2 N3 N4 N5 ACTIN MSATH3K4me3 relative enrichment Nanog promoter sites Non-silencingshHMGN2-381Repression of Oct4 and Nanog in Hmgn2 knockdown cells is accompanied by increases in the repressive mark H3K27me3Repression of Oct4 and Nanog in Hmgn2 knockdown cells is accompanied by a reduction in the active mark H3K4me3Schematic representation of the main chromatin structuralelements of enhancers (Martinez de Paz and Ausio, 2015)

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Knockdown High Mobility Nucleosomal Binding Proteins 2 (HMGN2) Alter the Histone Modification H4K4me3 and H3K27me3 and Regulates Stem Cells Pluripotency

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