Gene Expression in Broiler Myoblasts (RNA-Seq)

Chicken meat production

8 100 Populatin (Billion) 90 7 Chicken production (Million tones) 80 6 70 5

60 (Billion)

(Million) 4 50

3 Tones

30 Populatin 2 20 1 10

0 0

1950 1958 1966 1952 1954 1956 1960 1962 1964 1968 1970 1972 1974 1976 1978 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010

Earth Policy Institute Chicken breedings in Japan

名古屋コーチン比内地鶏 Broiler Import 100%!

長州黒かしわ

天草大王

御殿地鶏

http://www.j-chicken.jp/anshin/guide.html#place6 Layer and broiler chickens

Layer (e.g., White Leghorn) Broiler (e.g., UK Chunky) • For egg production • For meat production • Laying 280 eggs/year • Rapid growth • Weight: 2.8~3.4 kg • Weight: 4~5 kg Skeletal muscle tissue

A myofiber

Skeletal muscle tissue

Actomyosin filaments Skeletal muscle development & growth

Myoblast Myocyte Myotube

Differentiation Cell fusion Muscle formation

Activation Proliferation

Stem cell

Myofiber Myogenic differentiation of chick myoblasts

Day 0 Day 2 Day 4

Phase

DAPI

/ MHC

Takaya, Anim Sci J, 2017 Comparison between layer and broiler myoblasts

WL (layer) UKC (broiler)

Myoblast Myoblast

Compare growth, differentiation, and gene expression Growth of layer and broiler myoblasts

UKC WL 50 **

cells) 40

10 × ( 30 **

20 *

10 No. of myoblasts No. 0 0 24 48 72 96 Culture (h)

2.5×104 cells/well, GM, n = 3, *p < 0.05, **p < 0.01 Cell proliferation of layer and broiler myoblasts

EdU / DAPI

60 *

50 WL

40 (%)

cells +

20 EdU

10 UKC

0 WL UKC

1×105 cells/dish, GM, n = 4, *p < 0.05 Differentiation of layer and broiler myoblasts

MHC / DAPI (day 2)

WL UKC 50

WL 40

(%) 30 cells

+ 20 ** MHC

10 UKC

0 Day 0 Day 1 Day 2

1×105 cells/dish, DM, n = 4, **p < 0.01 Cell characteristics of broiler myoblasts

Myoblast Myocyte Myotube

Differentiation Cell fusion

Proliferation Gene expression patterns in chick myoblasts

Myoblast Myocyte Myotube Day 0 Day 1 Day 2

Gene expression RNA sequencing RNA sequencing of chicken myoblasts

WL (layer) UKC (broiler) Genome 1.2 billion bp 20 K genes

DNA

Transcription

RNA

Translation

Protein RNA sequencing

Day 0 Day 1 Day 2 WL ×3 ×3 ×3 UKC ×3 ×3 ×3

Total RNA isolataion ↓ RNA library preparation ↓ RNA-sequence

23.3 M reads/sample obtained 22.6 M reads/sample mapped to genome (97.2%)

46,389 transcripts identified 26,640 genes quantified

RNA-seq by NODAI Genome Research Center Differentially expressed genes (DEGs); UKC vs WL

1,032/26,640 (3.9%) Day 1 genes were differentially expressed between UKC and WL. 80

80 112

336

195 200

29 Day 0 Day 2

RPKM, |fold-change| ≥ 2, FDR p < 0.05 Ontologies of 336 DEGs (81 genes)

Gene ontology p Genes Proteinaceous NOV, HAPLN1, COL9A2, COL14A1, CRISPLD2, SMOC1, LUM, 7.1E-6 extracellular matrix ELN, COL6A3, FBN3, ECM2, COL16A1, SLIT3 COL14A1, COL21A1, COMP, F3, MMP27, MMP17, FBN3, TGM4, Extracellular matrix 3.4E-5 AGRN, FBN2 FGF19, BGLAP, CCK, ENPP2, C1R, ESM1, PTGFR, ADCYAP1, Extracellular region 7.7E-5 NOV, PGR, DNASE1, DKK3, BMPER, CXCL14, AGRN, NRG1, CD200R1 ENPP2, PDGFA, LUM, FAM132A, LRRC4C, NRN1, GREM2, ABI3BP, C1QTNF5, COMP, COL6A3, SERPINB10, PTN, NRG1, Extracellular space 3.6E-4 LOC422654, ECM2, SLIT3, PTHLH, GKN2, BMPER, COL14A1, F3, SEMA4D, IL12B, LIPC Axon 0.003 ALCAM, PTPRK, CCK, PVALB, STMN2, CHRNA7, AGRN, GAP43 Neuron projection 0.003 STMN2, TENM2, MAP2, AGRN, GHSR, GAP43, TPH2, ADCYAP1 SLC8A3, LPPR1, FLT4, TRPV3, PTGFR, APCDD1, KDR, ALCAM, Integral component 0.010 SEMA6A, THBD, SLC24A4, SLC6A7, SLC24A2, TENM2, of plasma membrane ADRA1B, CLDN1, TBXA2R, SEMA4D, NRG1, FAM26F Collagen trimer 0.027 COL9A2, C1QTNF5, COL14A1, COL6A3 PTPRK, THBD, CAPN5, CLSTN2, PDGFA, F3, BF2, ITGB5, GHSR, Cell surface 0.039 MDK

GOTERM_CC_DIRECT, p < 0.05 The 336 DEGs as surface markers of myoblasts

Major members of the 336 DEGs differentially expressed expressed between layer WL and broiler UKC myoblasts were membrane or extracellular proteins.

WL UKC

These genes may be useful as the markers for selective breeding of the chickens for meat production.

Nihashi, Sci Rep, 2019 Further journey for gene exploration Cell characteristics of broiler myoblasts

Myoblast Myocyte Myotube

Differentiation Cell fusion ↑???

Proliferation ↑??? Gene expression shift during differentiation

Day 0→1 Day 1→2 Day 0→2

value) p

WL (

log -

log2(fold-change)

UKC

Volcano plot by Dr Koji UMEZAWA 840 DEGs during myogenic differentiation

0 A1

-1

-2

B1 B2 d0 d1 d2 d0 d1 d2 WL UKC WL UKC

Generated by Heatmapper, RPKM, |fold-change| ≥ 4, FDR p < 0.05 Gene ontologies of 840 DEGs

A1 A2 B1 B2 Others A2 18

16 A2: Muscle-related B1 B1: Cell cycle 14 WL UKC

6 No. of genes No.

GOTERM_BP_DIRECT, p < 0.05 Protein-protein interaction (A1, 270 genes)

WL UKC

・ Collagen triple helix repeat

Generated by STRING Protein-protein interaction (A2, 393 genes)

WL UKC

・ Muscle contraction ・ Regulation of muscle contraction ・ Regulation of system process ・ Striated muscle contraction ・ Regulation of multicellular organismal process

Generated by STRING Protein-protein interaction (B1, 117 genes)

WL UKC

・ Cell cycle process ・ Cell cycle ・ Chromosome organization ・ Mitotic nuclear division ・ Organelle organization ・ Mitotic cell cycle ・ Cell division ・ Single-organism organelle organization ・ DNA conformation change ・ DNA integrity checkpoint ・ Cellular response to DNA damage stimulus

Generated by STRING Protein-protein interaction (B2, 45 genes)

WL UKC

No functional cluster

Generated by STRING PCA: Principal component analysis (主成分分析)

Primary PC

Secondary PC Expression in WL Expression level

Expression level in UKC PCA: Principal component analysis (主成分分析)

2000

WL day 0 WL day 1 0

PC2 WL day 2 UKC day 0 UKC day 1 UKC day 2

-2000 -5000 0 5000 PC1

Calculated by Dr Koji UMEZAWA PCA: Principal component analysis (主成分分析)

PC1 (Differentiation)

PC2 (Breed)

Calculated by Dr Koji UMEZAWA PENK: pro-enkephalin

Tyr-Gly-Gly-Phe-Met: Met-enkephalin (MENK) Tyr-Gly-Gly-Phe-Leu: Leu-enkephalin (LENK) What’s enkephalin

Enkephalin is one of opioid peptides.

Enkephalin Emotion Endorphin Analgesia Dynorphin Appetite

Brain Muscle narcotic narcotic? Enkephalins suppress myoblast proliferation

Control MENK LENK 30

cells) ** 4 **

× (

5 No. of myoblasts No. of myoblasts

0 0 24 48 72 Enkephalin treatment (h)

2.5×104 cells/well, GM, n = 4, **p < 0.01 Enkephalin suppresses myoblast proliferation

Enkephalin Myoblasts Myocytes

Differentiation

Proliferation

Why does enkephalin suppress myoblast growth?

I (anybody) don’t know! It’s science.

Nihashi, Sci Rep, 2019 Opioid peptides in foods

Milk Spinach Rice

Albumin, casein, gluten, lactoferrin, etc.

Opioid peptides National Institute of Infectious Diseases, Japan Evolution of Chicken Breeds

Gallus gallus

Layer Broiler Gene expression in broiler myoblasts (RNA-seq)

Myoblast Myocyte Myotube

Differentiation Cell fusion ↑Muscle genes

Proliferation ↑Cell cycle genes ? 43 ERVKs are detected in chicken myoblasts

Gene Product Gene Product Gene Product

LOC107052718 ERVK-11 Pol protein LOC107053122 ERVK-18 Pol protein-like LOC107049272 ERVK-8 Gag polyprotein-like

LOC107054241 ERVK-25 Pol protein-like LOC107053216 ERVK-113 Pol protein-like LOC107049274 ERVK-18 Pol protein-like

LOC107054837 ERVK-18 Pol protein-like LOC107056268 ERVK-18 Pol protein-like LOC107049277 ERVK-9 Pol protein-like

LOC107050425 ERVK-25 Pol protein-like LOC107053591 ERVK-18 Pol protein-like LOC107049276 ERVK-18 Pol protein-like

LOC107050867 ERVK-18 Pol protein-like LOC107056419 ERVK-18 Pol protein-like LOC107051888 ERVK-25 Pol protein-like

LOC107051645 ERVK-18 Pol protein-like LOC107054036 ERVK-9 Pol protein-like LOC107051891 ERVK-8 Pol protein-like

LOC107051640 ERVK-113 Pol protein-like LOC107057172 ERVK-18 Pol protein-like LOC107052380 ERVK-25 Pol protein-like

LOC107055731 ERVK-25 Pol protein-like LOC107057173 ERVK-8 Gag polyprotein-like LOC107049326 ERVK-18 Pol protein-like

LOC770574 ERVK-25 Pol protein LOC107055329 ERVK-18 Pol protein-like LOC107049332 ERVK-113 Pol protein-like

LOC107052504 ERVK-18 Pol protein-like LOC107049264 ERVK-8 Gag polyprotein-like LOC107049350 ERVK-25 Pol protein-like

LOC107052568 ERVK-113 Gag polyprotein-like LOC107055436 ERVK-8 Pol protein-like LOC107049474 ERVK-9 Pol protein-like

LOC107051673 ERVK-18 Pol protein-like LOC107055437 ERVK-18 Pol protein-like

LOC107052661 ERVL-18 Pol protein-like LOC107055442 ERVK-18 Pol protein-like

LOC107052685 ERVK-11 Pol protein-like LOC107055443 ERVK-25 Pol protein-like

LOC107052830 ERVK-113 Pol protein-like LOC107055445 ERVK-25 Pol protein-like

LOC107052829 ERVK-113 Pol protein-like LOC107049273 ERVK-9 Pol protein-like ERVK: Endogenous retrovirus K member

RNA retrovirus

ERV

Denner, Clin Microbiol Rev, 2012 ERVs in chicken genome

ERVL ERVK

ERVs occupy > 21 Mb (~2%) of chicken genome!

Barr, J Virol, 2005 ERVKs and their neighboring genes

10 No. of genes of No.

90 10 20 30 40 50 60 70 80

-30 -90 -80 -70 -60 -50 -40 -20 -10

100

-100 Overlap Distance from ERVK (kb)

ERVK should be randomly inserted into the genome. However, there is a lot of genes existing near the transcribed ERVKs.

The ERVKs having neighboring genes are transcribed. ERVK regulates its neighboring gene expression

? ERVK

Do ERVK transcriptions affect the expression of neighboring genes in myoblasts? Highly-transcribed ERVKs in broiler myoblasts

Myoblast Myocyte Myotube

BrK1

BrK2

▭ Layer ▬ Broiler BrK1 and its neighboring genes

BrK1 FAM19A2

LOC107052719 Chr 1 10 kb

▭ Layer ▬ Broiler

60 120 0.12 * * * * 50 * 100 0.10

40 (RPKM) 80 * 0.08 (RPKM)

30 60 0.06 (RPKM) **

20 40 0.04 BrK1

10 20 FAM19A2 0.02 LOC107052719 0 0 0.00 Day 0 Day 1 Day 2 Day 0 Day 1 Day 2 Day 0 Day 1 Day 2 BrK2 and its neighboring genes

SLC1A7 SCP2 BrK2 FAM159A ORC1 PODN LOC772243 ZCCHC11 ECHDC2 ZYG11B GPX7 PRPF38A LOC107054037 COA7 MIR6549 Chr 8 10 kb

▭ Layer ▬ Broiler

8 1.2

7 * * * 1.0 6 0.8

5 (RPKM)

4 0.6 (RPKM)

3 * 0.4 **

BrK2 ** 2

LOC772243 0.2 1

0 0.0 Day 0 Day 1 Day 2 Day 0 Day 1 Day 2 ERVK may affect neighboring gene expression

The 43 ERVK transcriptions were identified in chicken myoblasts.

22/43 of the ERVKs were differentially expressed in between layer and broiler myoblasts.

These ERVKs may affect the expression levels of their neighboring genes.

Virally introduced ERVKs into the genome may be involved in animal phenotype.

Takaya, in revision Hypothesis

ERVK ↑Differentiation ERVK ERVK ↑Cell cycle Gallus

Layer Broiler