mxc mutant alleles:

Lethal (L1-L2)Pharate lethal

viable

Drosophila spermatogenesis

mxc mutations affect germ cell developmentmxc mutations causes loss of germline stem cells (GSCs), disruption of transit amplification divisions, and premature differentiation:

mxc mutations affect histone biosynthesis

mxc mutations affect the DNA repair response following replicative stress

multi sex combs (mxc) controls histone biosynthesis and replicative stress response in Drosophila

Severine Landais and Leanne Jones, Salk Institute, San Diego, CA, USA.

H1 H1

10 mxcG46;Nanos-Gal4>Gal4RNAi, 5do

mxcG46;Nanos-Gal4>H3RNAi, 5do

DAPITjVasaDE-cad

Hub

0

2

4

6

8

0

10

20

30

40

50

Num

ber o

f cel

l EdU

+/te

stis

Cystcells

Germcells

mxcG46

Control

Num

ber o

f GSC

s Ed

U+/

testi

s

P<0.0001

P<0.0001P<0.0001

mxcG46 testes

U7EY11305 testes

Fold

cha

nge

rela

tive

to c

ontr

ol

lok(Chk2)

mei-41(ATR)

tefu(ATM)

mus304(ATRIP)

Control mxcG4620 20

Control Controlhydroxyurea

mxcG46

mxcG46;Nanos-Gal4>H3RNAi

DAPIpgH2Av

pgH2Av

10

Edu incorporation is affected in mxc mutant testes

The number of cells EdU+ decreases in mxc mutant testes

Phosphorylated gH2Av accumulates in mxc mutant germ cells Genes that mediate the DNA repair response are downregulated in mxc mutant testes

ABSTRACT

The multi sex combs (mxc) gene as been characterized as a polycomb group gene more than 20 years ago. The strongest mutant alleles of mxc are lethal early in development, whereas the weakest, viable alleles (mxcG43 and mxcG46) are sterile. We characterized mxc mutant phenotype in the male gonad and found that spermatogenesis was disrupted at the earliest stages of germ cell development, with incomplete rounds of transit amplification divisionsand loss of germline stem cells (GSCs). We and others found that Mxc is a critical factor for the histone locus body (HLB) assembly, and proper HLB formation is impaired in mxc mutant cells, although all the core histone mRNA levels are not severely decreased in several mxc mutant alleles, prior to a general decrease of core histone mRNA. On the contrary, histone H3 mRNA is greatly increased in mxcG46. Interestingly, a specific depletion of H1 protein was detected in mxc mutant germ cells only, and H1 RNAi-mediated knockdown causes GSCs loss, but fail to recapitulate the overall germline defects characteristic of mxc mutation. On the other hand, H3 RNAi specifically expressed in the germline rescues 100% of mxcG46 germline phenotype. This suggests collaborative effects of impaired histones production on germ cells maintenance and proliferation. Accordingly, we observe that mxc mutant testes fail to incorporate EdU properly, which suggests replicative stress, a hallmark of histone level defects. A concomitant accumulation of phosphorylated gH2Av (pgH2Av) is observed in germ cells. However, pgH2Av persists in mxc mutant germ cells, suggesting a failure of the DNA repair response. We found that genes critical to this response were down regulated in mxc mutant testes, suggesting a role for Mxc in DNA repair. Therefore, mxc appears as a critical factor for chromatin maintenance involved in both the synthesis of basic packaging components and the DNA repair machinery.

Mxc colocalizes with core components of the HLB

HLB assembly is impairedIn mxc mutants.(Described in White et al,JCB, Vol. 193 (4) 2011.

Impaired histone mRNA levels in mxc mutants

Decompaction of polytene chromosomesIn mxcG43 salivary glands

Germline specific H1 protein depletion in mxc mutants gonads

Control larvae

mxcG43 larvae

Control adult

mxcG43 adult

Loss of GSCs after 10 days of H1 RNAi expression in the germline

Rescue of mxc mutant germline phenotype with germline specific expression of H3 RNAi

H1 RNAi expressed in the germ cellsFail to recapitulate mxc mutant phenotype

Cell autonomous effect of mxc mutation on GSCs maintenance

Control mxcG46 rescue

Controllarvae

mxcG46

larvaemxcG43

larvae

Controladult

mxcG46

adultmxcG43

adult

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