Cerebrum, Liver, and Muscle Regulatory Networks Uncover Maternal Nutrition Effects in Developmental Programming of Beef Cattle During Early Pregnancy

Cerebrum, liver, and muscle regulatory networks uncover maternal nutrition effects in developmental programming of beef cattle during early pregnancy

Wellison J. S. Diniz1*, Matthew S. Crouse2, Robert A. Cushman2, Kyle J. McLean3, Joel S. Caton1, Carl R. Dahlen1, Lawrence P. Reynolds1, Alison K. Ward1

1Department of Animal Sciences, North Dakota State University, Fargo. 2USDA, ARS, U.S. Meat Animal Research Center, Clay Center, NE. 3Department of Animal Science, University of Tennessee, Knoxville, TN *Corresponding author: [email protected]

SUPPLEMENTARY FIGURES

Figure S1. Saturation curve of the cerebrum (A), liver (B), and muscle (C) RNA-Seq data. The plot shows the number of detected genes represented as full dots (y-axis) and the new detections per each million of additional reads sequenced (x- axis) at increasing sequencing depths for different samples after removing the lowly expressed genes. The plot was created using NOISeq v.2.26.0 on R environment.

Figure S2. PCA analysis from RNA-Seq data of 17,164 genes across three tissues and 42 samples. Each dot represents one tissue from one animal. The plot was created using DESeq2 v.1.22.1 on R environment.

A B

Figure S3. Venn diagram of 1,160 unique genes used for tissue-to-tissue network construction overlapped by tissue (A) and category (B). The genes from cerebrum (n = 228), liver (n = 672), and muscle (n = 325) were prioritized considering the following criteria: (1) differentially expressed between RES and CON groups; (2) tissue-specific; and (3) key TFs based on RIF1 or RIF2. Venn diagram was created using Venny v.2.1. (https://rb.gy/jxxufy).

TREH

GIPC2

PFKFB1 SLC2A2 ZNF500 ZNF568 MLXIPL B3GNT8 ENSBTAG00000053893 ABCA4 ADM2 E2F1 HSD17B14 LY6G6F CYP11A1 F13B SLC22A14 CNBP ENSBTAG00000038461 BAIAP2L2 ECM1 ALDH1L1 CAPN8 KRT8 SEPTIN4 TOP1MTENSBTAG00000038549 CCL16 PTPRR ANKRD35 MS4A7 ENSBTAG00000003289 CD300H WFDC2 MAP1B ENSBTAG00000054798 CYP8B1 CFD SERPINA1 G6PC3 ENSBTAG00000049683MCOLN2 ENSBTAG00000000336 FARP2 HNF1B MTFP1 HBE4 LCN2 ENSBTAG00000050476 SSX5 PRR5 TSKS SLC23A3 DEFB13 NGP CATHL2 FOXM1 BLK IRF3 CEBPEENSBTAG00000023648 ENSBTAG00000054525CCDC42 IRF8 CPS1bta-mir-451APOC3 STAB2 ENSBTAG00000050563 SLC38A3 GATA1 MOSPD3 STMN4 LY6G6D SLC51BENSBTAG00000009364 E2F4 PRSS57 ANGPTL3 GLUD1 ZNF184 CAMP ZKSCAN2 ENTPD8 SLC16A11 MGAM ENSBTAG00000015987ENSBTAG00000013345AADAC URAD GRAP2ENSBTAG00000039196APOC2 PUS1 MASP2 TPRA1 SLC10A1 MT1A ENSBTAG00000049257 C5 PROZ IGSF5 SLFN14 USF2 IGFBP1 ENSBTAG00000030683PIK3C2G S100A9 ENSBTAG00000023411 AGXT GC ENSBTAG00000026917 PGLYRP2 DPYSL3 ENSBTAG00000053931 MT2A TBX10 GCGR PALM3 ENSBTAG00000054524 STAP1 DEDD2 RBP5 CLDN4 ZNF713 ABCA2 GOLT1A FMO3 GYPA VTN HP NUPR1 ENSBTAG00000034657S100A12 KEL SFTPB GATA5 ENSBTAG00000040409 NAGS SPN ENSBTAG00000047621 KNG1 PNPLA6 HNF1A KMO KCNN4 ENSBTAG00000053783ENSBTAG00000023666 S100G TBC1D10C CYP4A11 DUOX2 AFM SLC22A7 C29H11orf86 GTF2IRD1 HRG CYP4F2 SLC5A4 GLB1L3 MIOX ALDH8A1 GSTA1 HSD3B1 AFP LIPC CD27 ENSBTAG00000038058 CIAO3 ENSBTAG00000038171 AOC1 SPDYC DEFB7 CABP2 MPO IDO2 S100A8 ZNF423 RETN CLEC1B HPN IYD UOX PLEK F10 THEG ENSBTAG00000024901SLC22A9 HAL F11 F2RL3 SERPIND1 OSM CHIA DZIP1 GATA4 NAALADL1 NMRAL1 FABP1 APOC4 SMAD4 GYS2 ACSM5 ENSBTAG00000050209 HPD ZNF383 ENSBTAG00000047175ENSBTAG00000015557 ENSBTAG00000030297IKZF1 VNN1 MPL TREML2 APOA2 CGN1 AQP8SERPINA3-3 ENSBTAG00000051856 HAAO OXER1 SLC17A3 ENSBTAG00000030416 CCRL2 APON CTRCENSBTAG00000052022 CIDEC SERPINA11 SLCO1B3 TUBB1 ENSBTAG00000055241 SLC22A18 bta-mir-122 ENSBTAG00000049782 ART4 GLTPD2 ENSBTAG00000052808 GIMAP4 CHST13 FXYD4 APOH PRODH2 ACE2 NR1H4 ATP8B3 FOXA3 ADTRP FGA F9 OXT ENSBTAG00000001297 ENSBTAG00000049436SERPINF2 SERPINA7 NR1I2 ROS1 SEC14L4 A1BG UGT1A1 IL20RA IGSF23 ETNPPL DPYS ENSBTAG00000011961 ENSBTAG00000014220HSF2 GLYAT HGD SPP2 PTPN7 IL12RB1 KYNU HPX LYL1 GDF2 MGC137211 GP1BA F5 C15H11orf52 LRG1 TMEM184A ASCL2 HAMP SMCO3 CPN2 AGMAT ENSBTAG00000052659 NR1H2 AGT GSTA2 CL43 TF BHLHE41 PTI ZNF521 MBL1 MARCO C8A ASGR1 ENSBTAG00000054649 CREB3L3 ENSBTAG0000001198520ALPHA-HSD RFX7 SERPINA3-8 APOA4 HSF4 HMGCS2 ENSBTAG00000054844 SERPINA3-1PLA2G1B TDO2 CDH16 LBP UPB1 ENSBTAG00000054314 ENSBTAG00000023939 ENSBTAG00000015139 C8B ACMSD FGG KLF1 SPTA1 TMPRSS6 BHMT HABP2 MT1E ENSBTAG00000054975INHBE TRIM58 HOGA1 FCGR2A ENSBTAG00000012192 ENSBTAG00000039337 SERPINA6 ENSBTAG00000040034 CATHL1 NR0B2 CD22 AKR1D1 ITIH3 ALB SMIM6 P2RX1 SOAT2ENSBTAG00000010155 SULT1C4 C1QTNF12 SLC17A1 HKDC1 SGK2 APOB HSPA1A ALAS2 SUSD2 DAO KLB ENSBTAG00000052514 TMEM140 OTC GYPB ASGR2 CD209 H2BC11 ENSBTAG00000053177 RAB17 ARG1 SERPINA3-7ENSBTAG00000037509 HAO2 TM4SF5 RGS18 FTCD GJB1 CCR3 ABCC2 TM4SF4 PLA2G12B ICAM5 MGC137014ITIH2 SLC17A2 RAD21L1 TRIM10 ENSBTAG00000049953 SOX6 LTF GP9 SLC5A9 ENSBTAG00000014857 KLKB1 HNF4A ITIH1 EBF1 HSD17B2 F12 GBP6 AGXT2 RHAG RXRG ENSBTAG00000051797 F7 LECT2 PLG ENSBTAG00000022715 GFI1B ICAM4 ACSM3 FETUB TMEM150B CLEC4F MAP3K15 AHSG SPIC SLC23A1 MYL6B ENSBTAG00000054604 TREML1 ZBTB32 ENSBTAG00000047356 GBP4 CPB2

ENSBTAG00000038655 ENSBTAG00000005609 SERPINA10 ENSBTAG00000000229 PBLD

Figure S4. Gene co-expression sub-network with 427 nodes and 81,308 interactions from the first neighbors of the GATA1, HSF2, and PLEK2 transcription factors. Only nodes with a correlation greater than |0.9| are shown. Nodes are colored considering their tissue (cerebrum, liver, and muscle). Overlapped genes between analysis were colored based on the tissue with its maximum expression. Transcription factors are represented by a diamond shape. Gene network was created on Cytoscape v.3.7.

HES6 RCOR3 0/- FOXO4 0/- 0/+ HOXA7 -/+ MYF6 0/+ 0/+ 0/- NANOS1 0/- -/+ 0/+

ZBTB18 0/+ SMAD4 ZBTB33 0/- MYOD1 0/+

+/- 0/+ +/- 0/- 0/- ## 0/+ +/+ +/-

CDKN1A 0/- MYOG 0/- +/- ZNF131 IGSF10

+/+ 0/- 0/+ 0/+ 0/+ 0/- 0/- MSTN

PAX7 MYF5

Figure S5. Gene co-expression network of myogenic factors. Differentially expressed nodes are labeled in pink. Differentially connected nodes have green borders. Transcription factors are represented by a diamond shape. Single line edges are based on the differential gene co-expression analysis, whereas the double lines are based on the tissue condition-specific network (PCIT). Red and blue lines represent negative or positive correlation between CON and RES groups, respectively. ##based on Robinson et al. (2017, Doi: 10.1016/j.bbrc.2017.09.007). Gene network was created on Cytoscape v.3.7.

Ribosome

Complement and coagulation cascades 40 SUMOylation of intracellular receptors

Cell cycle Ribosome biogenesis in eukaryotes

Cholesterol metabolism 30

Maturity onset diabetes of the young

Pertussis

Basal transcription factors 20 Fanconi anemia pathway

Glycosaminoglycan degradation Tryptophan metabolism 10 Starch and sucrose metabolism

Arginine biosynthesis

Staphylococcus aureus infection 0 DNA replication

Ubiquitin mediated proteolysis

Neuronal System

Protein processing in endoplasmic reticulum Ras activation upon Ca2+ influx through NMDA receptor

Transport of inorganic cations/anions and amino acids/oligopeptides

GABA synthesis, release, reuptake and degradation Potassium Channels

Neurotransmitter receptors and postsynaptic signal transmission Neurotransmitter release cycle

p53 signaling pathway

GABAergic synapse mTOR signaling pathway Epstein−Barr virus infection Transmission across Chemical Synapses Dopaminergic synapse Retrograde endocannabinoid signaling Acute myeloid leukemia Porphyrin and chlorophyll metabolism Insulin signaling pathway Measles Glycosylphosphatidylinositol(GPI)−anchor biosynthesis Signaling pathways regulating pluripotency of stem cells HIF−1 signaling pathway Ras signaling pathway Focal adhesion Alzheimer's disease PI3K−Akt signaling pathway Non−alcoholic fatty liver disease (NAFLD) Metabolic pathways Oxidative phosphorylation Rap1 signaling pathway Chronic myeloid leukemia Proteoglycans in cancer Huntington's disease Pathways in cancer MicroRNAs in cancer Parkinson's disease Muscle contraction MAPK signaling pathway Regulation of actin cytoskeleton Striated Muscle Contraction Endometrial cancer

Non−small cell lung cancer Cardiac muscle contraction

HTLV−I infection Melanoma Neurotrophin signaling pathway Oxytocin signaling pathway Endocytosis Arrhythmogenic right ventricular cardiomyopathy (ARVC) Prostate cancer Axon guidance FoxO signaling pathway Adrenergic signaling in cardiomyocytes Dilated cardiomyopathy Muscle Liver Cerebrum

Figure S6. Heat map of over-represented signaling pathways of co-expressed genes from the cerebrum, liver, and muscle tissues. The matrix is color-coded based on the –log10 (adjusted p- value) according to the legend. Heatmap was constructed using pheatmap v.1.010. on R.