Investigation of regulators of hippocampus functions and adult neurogenesis by system biology approaches Haifang Wang July 7, 2015

Overview circadian clock Post-transcriptional regulation by miRNA Post-translational regulation by TIP60 (collaborate with Dr. Anastasia Repouskou) TIP60 function in the hippocampus (Collaborate with Dr. Inga Urban) Molecular signatures of progenitor cells in adult primate (Collaborate with Dr. Anton Tonchev)

Part 1. TIP60 function in the hippocampus

TIP60 is a well-known lysine accetyltransferase that with many known histone as well as non-histone substrates, and highly expressed in adult forebrain. It is function in the mammalian nervous system is largely unknown. Recent studies suggest that epigenetic mechanisms of gene expression such as histone acetylation play an important role in memory formation. Forebrain-specific Tip60 conditional knock-out (cKO) mice gained weight, got seizures and showed limb clasping and elevated nervosity. On molecular level several immediate-early genes, which have been implicated in learning and memory, are significantly up- regulated in the CA1 region of cKOs,. We want to elucidate the role of TIP60 in the mammalian hippocampus by whole transcriptome comparison of the WT and cKO mice. Background

TIP60 immunoreactivity in the hippocampus Tip60 f/f (6) CaMKCreER T2 Tip60 f/f (6) Tip60 f/f (4) CaMKCre Tip60 f/f (5) DAPI = red TIP60 = green Inga Urban Part 1. TIP60 function in the hippocampus CA1 CA2 CA3 DG CA1 CA2 CA3 DG CA1 CA2 CA3 DG CA1 CA2 CA3 DG

RNA-seq analysis pipeline Part 1. TIP60 function in the hippocampus

Result 1. A example: Npas4, up-regulated in both inducible and constitutive lines Part 1. TIP60 function in the hippocampus

Result 2. 2-dimensional principal component analysis (PCA) show a clear separation between different groups Part 1. TIP60 function in the brain

Result 3. Number of differential expressed genes (DEGs) using different log2-transformed fold change (log2FC) as cutoff log2FCcommoninducibleconstitutive Up-regulated Down-regulated Part 1. TIP60 function in the brain Significance of the overlap (|log2FC|>0.5): Fisher’s exact test p-value< 2.2e-16, odd ratio=18.6

Result 4.1. expression pattern of differential expressed genes in inducible lines Part 1. TIP60 function in the brain 983 down- regulated genes 868 up- regulated genes

Result 5. RNA-seq fold changes in inducible lines validated by qPCR Inga Urban Part 1. TIP60 function in the brain

Result 6.1. RNA-seq fold change validated by ISH: Npas4 (neuronal PAS domain protein 4) Tip60 f/f CaMKCreER T2 Tip60 f/f Inga Urban CA1

Result 6.2. RNA-seq fold change validated by ISH: Esr1 (estrogen receptor 1) Tip60 f/f CaMKCreER T2 Tip60 f/f Tip60 f/f CaMKCre Tip60 f/f Inga Urban CA1

Result 6.4. RNA-seq fold change validated by ISH: Lin7b (lin-7 homolog B) Inga Urban Part 1. TIP60 function in the brain a c Tip60 f/f CaMKCreER T2 Tip60 f/f CA1

Result 7. DEGs are enriched in certain biological function Part 1. TIP60 function in the brain

Result 8. The promoter of the down-regulated genes are enriched in binding site of TFs with neural function down-regulated genesup-regulated genes AP-2 HIC1E2F1 ZIC4E12 ZIC3NHLH1 HEN1 MTF1 NFKB CP2 INSM1 EBF1 PLAG1 MYOD E2F1 HINFP1 E12 E47 GLIS2 NHLH1 HEB SZF11 NRSF Part 1. TIP60 function in the brain

Result 9. Comparison with forebrain-specific Gcn5 conditional KO RNA-seq Part 1. TIP60 function in the brain Stilling RM, Ronicke R, Benito E, Urbanke H, Capece V, et al. (2014) K-Lysine acetyltransferase 2a regulates a hippocampal gene expression network linked to memory formation. EMBO J 33:

Part 2. Molecular signatures of progenitor cells in adult primate brain

Stem cells in primate brain Part 2. Molecular signatures of progenitor cells in adult primate brain Neurogenesis in the adult brain is restricted to the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) and the subventricular zone (SVZ) along the walls of the lateral ventricle.

Increased proliferation and neurogenesis after ischemia in monkeys (SVZa) CONTROL ISCHEMIA Phenotype Progenitor Neuron Glia Tonchev et al., 2003 Part 2. Molecular signatures of progenitor cells in adult primate brain

Primate-specific neuroanatomical features of SVZ Part 2. Molecular signatures of progenitor cells in adult primate brain Monika Chanu Chongtham

Tissue sampling and comparisons SVZa from Sham VS. ischmia DEGs in ischmia SVZa SVZi from Sham VS. ischmia DEGs in ischmia SVZi RNA- seq Common/diff erent DEGs Functional analysis, pathway analysis,, experimental validation…… Japanese monkeys Part 2. Molecular signatures of progenitor cells in adult primate brain

RNA-seq analysis pipeline

Result 1. A example: CLU, enhanced after ischemia in SVZa Part 2. Molecular signatures of neuronal stem cells

Result 2. summary of the differential expressed genes in SVZa and SVZi Part 2. Molecular signatures of neuronal stem cells SVZaSVZicommon Enhanced Decreased Significance of the overlap: Fisher’s exact test p-value< 2.2e-16, odd ratio=16.9

Result 3. neuron differentiation/development genes are enriched in up-regulated genes, while respiratory chain genes are enriched in down-regulated genes Part 2. Molecular signatures of neuronal stem cells

Tissue sampling and comparisons SVZa VS. Striatum DEGs in SVZa SVZi VS. CA1 DEGs in SVZi RNA- seq SVZa from Sham VS. ischmia DEGs in ischmia SVZa SVZi from Sham VS. ischmia DEGs in ischmia SVZi RNA- seq Common/diff erent DEGs Functional analysis, pathway analysis,, experimental validation…… German monkeysJapanese monkeys Part 2. Molecular signatures of progenitor cells in adult primate brain

Result 4. Comparison of DEGs in ischemia vs. sham and SVZa vs. striatum Ischmeia vs. sham SVZa vs. striatum common enhanced Decreased Part 2. Molecular signatures of progenitor cells in adult primate brain Significance of the overlapped enhanced genes: Fisher’s exact test p-value< 2.2e-16, odd ratio=5.1

CD38MARCH8FAM124AMROH8RRP8 CLIP2A2ML1FAM168AMVB12BSAMD4B BMPR1AAAED1FAM222A-AS1MYH6SCARA3 NLGN3ABCD3FGFR2NACC2SCD5 SP9ABHD17BFICDNCAM1SEMA6D GPR17ATF7FITM2NFICSF1 GREB1ATL3FNBP1NR4A3SLC25A48 MEX3DATP1A4FOXRED2OGFRL1SLC26A5 MAML3ATXN1LFRMD5PCDHA4SLC35E2 LFNGBBXFYCO1PCDHGA2SNORA80B RBPJBROXGALCPCDHGA3SNORD84 MAML2CCR6GNAO1PCDHGA4SPTLC1 DIP2ACORO1BGPM6BPCDHGB1TAB1 DOCK1CXorf38GPR173PCDHGC3TCP11L2 DUSP16DAZAP2GSTCDPCSK6THEM4 EFCAB14DCAF8HIP1PCYT1ATLN1 EHMT1DENND5AHMBOX1PFKFB2TMEM41B ELAVL3DESI1HNMTPGM2TMEM87A ELMO1ULK2HNRNPUL1PHACTR4TMTC2 ENTPD5WASF2IL4I1PHF12TRAF6 ETV3WDFY2IL6STPIN4P1TRIM14 LOC284837WWTR1IL9PMP2TRIM56 LOC285847ZNF281JAKMIP3POLR2ATRIOBP MAP7D3ZNF699KIF13APOU3F3TSPY26P MEGF10ZNF704KREMEN1PPFIA1TTLL11 RFX4ZSCAN12LIMS1PPM1KQKI RHPN2PXDC1LIX1LPTPLBREST Part 2. Molecular signatures of progenitor cells in adult primate brain

Result 5. CD38 expression pattern validated by ISH Sham SVZa Sham SVZi ischemic SVZa ischemic SVZi Corpus callosum Striatum SGZ DG SGZ Part 2. Molecular signatures of progenitor cells in adult primate brain B3B4

Future work More differential expressed genes will be validated by ISH. For the genes validated by ISH, in vivo loss- or gain-of- function experiments could provide valuable information on their possible function in neurogenesis. We observed that genes expression pattern are quite different for different subregions of SVZa, which indicate that they may have different cell types or neuronal functions, so next we will systematically search those genes and study their functions. Long non-conding RNAs (lncRNA) have been shown involved in neurogenesis recently, so we will also identify the differential expressed lncRNA after ischemia. Part 2. Molecular signatures of progenitor cells in adult primate brain

Result 3.2. expression pattern of common DEGs Part 1. TIP60 function in the brain

Neurogenesis in the adult brain is restricted to the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) and the subventricular zone (SVZ) along the walls of the lateral ventricle. It has been shown that brain ischemia in macaque enhanced the proliferation of neural progenitor cells in SVZ, while little is known about the molecular mechanisms. Here we applied RNA-seq on monkey SVZ samples, combined with high-throughput ISH, to identify the molecular signatures of primate progenitor cells on genome- wide basis before or after brain ischemia. Part 2. Molecular signatures of progenitor cells in adult primate brain Background

Result 6.3. RNA-seq fold change validated by ISH: Syt7 ( synaptotagmin VII ) Tip60 f/f CaMKCreER T2 Tip60 f/f Inga Urban

Future work In order to further elucidate the role of TIP60 in the hippocampus, the genes directly regulated by TIP60 have to be identified. So both ChIP-seq as well as co- immunoprecipitation experiments are planned in order to identify TIP60 binding sites and to shed further light on this direct targets.

Primate Model of Global Cerebral Ischemia (T. Yamashima, Kanazawa, Japan) Part 2. Molecular signatures of progenitor cells in adult primate brain Anton Tonchev