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

Investigation of regulators of hippocampus functions and adult neurogenesis by system biology approaches

Haifang Wang

July 7, 2015

Overview

circadian clockPost-transcriptional regulation by miRNAPost-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

Tip60f/f (6) CaMKCreERT2 Tip60f/f (6)

Tip60f/f (4) CaMKCre Tip60f/f (5)

DAPI = red TIP60 = green

Inga Urban


CA1

CA2

CA3DG

CA1CA2

CA3DG

CA1

CA2

CA3

DG

CA1

CA2

CA3DG

Step Description Tool/ScriptNon-default parameters

1 Quality Control FastQC -

2Mapping to reference

genome (GRCm37/mm9)

TopHat(v2.0.8) --no-coverage-search

3Read counting in

transcripts

bedtools (bedtools coverage)

-

4Detection of

differential gene expression

DESeq (R-package)

pairwise comparisons of

groups

5 Visualization IGV -

RNA-seq analysis pipeline


Result 1. A example: Npas4, up-regulated in both inducible and constitutive lines


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

　 log2FC common inducible constitutive

Up-regulated0 1520 2891 3172

0.5 594 868 2086

Down-regulated

0 2605 3601 3886

0.5 748 983 1917


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


983 down-regulated

genes

868 up-regulated

genes

Result 5. RNA-seq fold changes in inducible lines validated by qPCR

Inga Urban


Result 6.1. RNA-seq fold change validated by ISH: Npas4 (neuronal PAS domain protein 4)

Tip60f/f CaMKCreERT2 Tip60f/f

Inga Urban

CA1 CA1

Result 6.2. RNA-seq fold change validated by ISH: Esr1 (estrogen receptor 1)


Tip60f/f CaMKCre Tip60f/f

Inga Urban

CA1

CA1

CA1

CA1

Result 6.4. RNA-seq fold change validated by ISH: Lin7b (lin-7 homolog B)

Inga Urban


a c


CA1 CA1

Result 7. DEGs are enriched in certain biological function


　 Category Term PValue

down-regulated genes

GOTERM_BP_FAT cell adhesion 1.09E-06

GOTERM_BP_FAT ion transport 1.94E-06

GOTERM_BP_FAT intracellular signaling cascade 8.66E-06

KEGG_PATHWAY Gap junction 2.17E-05

KEGG_PATHWAY MAPK signaling pathway 2.65E-05

KEGG_PATHWAY Calcium signaling pathway 1.29E-04

KEGG_PATHWAY Focal adhesion 2.08E-04

GOTERM_BP_FAT neuron differentiation 2.29E-04

GOTERM_BP_FAT glycolysis 3.64E-04

GOTERM_BP_FAT cytoskeleton organization 4.25E-04

up-regulated genes

GOTERM_BP_FAT response to DNA damage stimulus 7.60E-08

GOTERM_BP_FAT cell cycle 2.47E-06

GOTERM_BP_FAT regulation of cell growth 2.19E-04

Result 8. The promoter of the down-regulated genes are enriched in binding site of TFs with neural function

down-regulated genes up-regulated genes

AP-2 AP-2HIC1 E2F1ZIC4 E12ZIC3 NHLH1HEN1MTF1NFKBCP2

INSM1EBF1

PLAG1MYODE2F1

HINFP1E12E47

GLIS2NHLH1

HEBSZF11

NRSF 　


Result 9. Comparison with forebrain-specific Gcn5 conditional KO RNA-seq

　 Tip60 Gcn5

up-regulated 868 28

down-regulated 983 10


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: 1912-1927

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

Stem cells in 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 ISCHEMIAPhenotypeProgenitorNeuronGlia

Tonchev et al., 2003


Primate-specific neuroanatomical features of SVZ


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

RNA-seq

Common/different DEGs

Functional analysis, pathway analysis,, experimental validation……

Japanese monkeys


Step Description Tool/Script Non-default parameters

1 Quality Control FastQC -

2Mapping to reference genome (RheMac2) TopHat(v2.0.8) --no-coverage-search

3Read counting in

transcriptsbedtools (bedtools

coverage) -

4Detection of differential

gene expression

DESeq (R-package) & paired

t-test

pairwise comparisons of groups (DEseq)

5 Visualization IGV -

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


　 SVZa SVZi common

Enhanced 577 282 136

Decreased 517 154 61

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


　 Category Term PValue

Enhanced in SVZa after ischemia


GOTERM_MF_FAT ion binding 2.27E-04

GOTERM_BP_FAT neuron differentiation 2.29E-04

GOTERM_BP_FAT neuron development 3.60E-04

GOTERM_CC_FAT anchoring junction 1.10E-03

KEGG_PATHWAY Gap junction 9.02E-03

Decreased in SVZa after ischemia

GOTERM_CC_FAT respiratory chain 2.16E-20

GOTERM_BP_FAT circulatory system process 1.62E-03

KEGG_PATHWAY Oxidative phosphorylation 2.78E-25

KEGG_PATHWAY Parkinson/Alzheimer 's disease 3.57E-23

Enhanced in SVZi after ischemia


GOTERM_MF_FAT ion binding 4.81E-04

GOTERM_MF_FAT cofactor binding 4.74E-03Decreased in SVZi

after ischemia GOTERM_BP_FAT electron transport chain 4.50E-02

Tissue sampling and comparisonsSVZa VS.

Striatum

DEGs in SVZa

SVZi VS. CA1

DEGs in SVZi

RNA-seq

RNA-seq

SVZa from Sham VS. ischmia

DEGs in ischmia SVZa

SVZi from Sham VS. ischmia

DEGs in ischmia

SVZi

RNA-seq

RNA-seq



Functional analysis, pathway analysis,, experimental validation……

German monkeys

Japanese monkeys


Result 4. Comparison of DEGs in ischemia vs. sham and SVZa vs. striatum

　 Ischmeia vs. sham

SVZa vs. striatum

common

enhanced 577 1220 135

Decreased 517 670 16


Significance of the overlapped enhanced genes:Fisher’s exact test p-value< 2.2e-16, odd ratio=5.1

CD38 MARCH8 FAM124A MROH8 RRP8CLIP2 A2ML1 FAM168A MVB12B SAMD4B

BMPR1A AAED1 FAM222A-AS1 MYH6 SCARA3NLGN3 ABCD3 FGFR2 NACC2 SCD5

SP9 ABHD17B FICD NCAM1 SEMA6DGPR17 ATF7 FITM2 NFIC SF1GREB1 ATL3 FNBP1 NR4A3 SLC25A48MEX3D ATP1A4 FOXRED2 OGFRL1 SLC26A5MAML3 ATXN1L FRMD5 PCDHA4 SLC35E2

LFNG BBX FYCO1 PCDHGA2 SNORA80BRBPJ BROX GALC PCDHGA3 SNORD84

MAML2 CCR6 GNAO1 PCDHGA4 SPTLC1DIP2A CORO1B GPM6B PCDHGB1 TAB1DOCK1 CXorf38 GPR173 PCDHGC3 TCP11L2DUSP16 DAZAP2 GSTCD PCSK6 THEM4

EFCAB14 DCAF8 HIP1 PCYT1A TLN1EHMT1 DENND5A HMBOX1 PFKFB2 TMEM41BELAVL3 DESI1 HNMT PGM2 TMEM87AELMO1 ULK2 HNRNPUL1 PHACTR4 TMTC2ENTPD5 WASF2 IL4I1 PHF12 TRAF6

ETV3 WDFY2 IL6ST PIN4P1 TRIM14LOC284837 WWTR1 IL9 PMP2 TRIM56LOC285847 ZNF281 JAKMIP3 POLR2A TRIOBP

MAP7D3 ZNF699 KIF13A POU3F3 TSPY26PMEGF10 ZNF704 KREMEN1 PPFIA1 TTLL11

RFX4 ZSCAN12 LIMS1 PPM1K QKIRHPN2 PXDC1 LIX1L PTPLB REST


Result 5. CD38 expression pattern validated by ISH

Sham SVZa

Sham SVZi

ischemic SVZa

ischemic SVZi

Corpus callosum

Corpus callosum

Striatum Striatum

SGZ

DG

DG

SGZ


B3 B4

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.


Thank you

Result 6.3. RNA-seq fold change validated by ISH: Syt7 (synaptotagmin VII)


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)


Anton Tonchev

Documents

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