Functions  of  the  CHD  family

complex,   the   dMep-­‐1   containing   complex   (dMec),   is   composed  of  only  two  subunits:  dMi-­‐2  and  dMep-­‐1  (figure   14).   Noteworthy,   a   Mec   complex   has   also   been   found   recently   in   Caenorhabditis   elegans   (Passannante   et   al.  

(2010)  PLoS  One).  It  is  actually  the  major  Mi-­‐2  containing   complex  in  Drosophila  and  during  the  early  development   of  C.  elegans.    

Figure   14   The   Drosophila   dMi-­2   containing   complexes.  

dNuRD:   Drosophila   nucleosome   remodeling   and   histone   deacetylase.   dMec:   Drosophila   Mep-­‐1   containing   complex.  

Adapted  from  Bouazoune  and  Brehm  (2006)  Chromosome  Res.  

2.5.2  Functions  of  the  CHD  family  

(1998)   Mol   Cell;   Zhang  et   al.   (1998)   Cell;   Wade  et   al.  

(1998)  Curr  Biol).    

 However,  recent  observations  propose  a  possible   relation   between   gene   activation   and   CHD   remodelers   (Miccio  et  al.  (2010)  Embo  J;  Murawska  et  al.  (2011)  PLoS   Genet;   Mathieu  et   al.   (2012)   Nucleic   Acids   Res).   It   has   been   shown   in   yeast   that   CHD   remodelers   can   regulate   genes   by   promoting   transcription   elongation   and   by   evicting   nucleosomes   located   at   promoters   (Walfridsson   et  al.  (2007)  Embo  J).  In  Drosophila,  every  CHD  chromatin   remodelers   (dCHD1,   dCHD3,   dMi-­‐2   and   Kismet)   co-­‐

localize   to   active   sites   of   transcription   on   polytene   chromosomes  (Marfella  and  Imbalzano  (2007)  Mutat  Res;  

Murawska  et  al.  (2008)  Mol  Cell  Biol;  Mathieu  et  al.  (2012)   Nucleic  Acids  Res).  Furthermore,  dCHD1  has  been  shown   to  interact  with  CTD-­‐2S-­‐P  and  elongating  factors,  whereas   Kismet   activity   seems   important   before   the   transition   to   the  elongation  step  (Srinivasan  et  al.  (2005)  Development;  

Lusser  et   al.   (2005)   Nat   Struct   Mol   Biol).   The   chromatin   remodelers   influences   on   gene   regulation   could   result   of   the  nucleosome  array  reorganisation,  the  restriction  of  the   DNA  accessibility,  the  removal  of  TFs  and  the  recruitment   of  chromatin  modifiers.  However,  their  exact  mechanisms   are  still  unknown.    

Nonetheless,   gene   regulation   is   not   the   only   function   attributed   to   the   CHD   chromatin   remodelers,   as   described  in  the  next  sections.  

 2.5.2.1  Chromatin  assembly  

  dCHD1   is   involved   in   chromatin   assembly   with   the   participation   of   the   Nucleosome   assembly   protein   1   (NAP-­‐1)  (Lusser  et  al.  (2005)  Nat  Struct  Mol  Biol)  and  it  is   involved  in  the  deposition  of  H3.3  in  the  male  pronucleus   (Konev  et  al.  (2007)  Science).  In  S.  pombe,  CHD1  loads  the  

H3  variant  CENP-­‐A  into  the  centromeres  and  it  is  required   for   proper   chromosome   segregation   (figure   12)   (Walfridsson  et  al.  (2005)  Nucleic  Acids  Res).  

2.5.2.2  DNA  replication    

In  yeast,  firings  from  the  origin  of  replication  are   inhibited   by   nucleosomes   (Simpson   (1990)   Nature).  

Chromatin  remodelers  are  needed  to  facilitate  the  access   of   DNA   polymerase   to   DNA.   The   CHD   family,   through   yCHD1,  is  involved  in  DNA  replication  in  cooperation  with   the   HMT   Set2   (Biswas  et   al.   (2008)   Genetics).   More   recently,   it   has   been   suggested   that   the   NuRD   complex   would  also  be  involved  in  DNA  replication.  It  is  based  on   an   observation   made   in   rapid   proliferating   lymphocytes,   where   NuRD   complexes   would   accumulate   in   pericentromeric   heterochromatin   of   chromosomes   1,   9   and   16,   during   S   phase   (Helbling   Chadwick  et   al.   (2009)   Chromosoma).  In  that  context,  NuRD  co-­‐localizes  with  the   active   replication   forks   and   the   Proliferating   cell   nuclear   antigen   (PCNA).   However,   dMi-­‐2   does   not   alter   the   DNA   replication  of  polytene  chromosomes  (Fasulo  et  al.  (2012)   PLoS  Genet)  and,  so  far,  the  function  of  Mi-­‐2  during  DNA   replication  is  still  poorly  understood.    

 2.5.2.3  DNA  repair  

  DNA   double   strand   breaks   (DSB)   can   result   of   a   stochastic   replication   failure,   an   exposition   to   reactive   oxygen  species  and  an  environmental  clastogens  (ionizing   radiation)  (Lobrich  and  Jeggo  (2007)  Nat  Rev  Cancer).  As   DSB   can   impair   genome   integrity   and   lead   to   tumorigenesis,   cells   induce   the   DNA   damage   response   (DDR)  to  arrest  the  cell  cycle  and  facilitate  the  DNA  repair   or   the   apoptosis   (Harper   and   Elledge   (2007)   Mol   Cell;  

Jackson  and  Barek  (2009)  Nature).  It  has  been  suggested   that   chromatin   remodelers   are   involved   in   DDR   (Clapier  

and   Cairns   (2009)   Annu   Rev   Biochem)   and   the   most   prominent   of   them   is   CHD4   (Polo  et   al.   (2010)   Embo   J;  

Urquhart  et  al.  (2011)  Genome  Integr;  Larsen  et  al.  (2010)   J   Cell   Biol;   Chou  et   al.   (2010)   Proc   Natl   Acad   Sci   USA;  

Smeenk  et  al.  (2010)  J  Cell  Biol).  The  CHD4,  and  thus  the   NuRD   complex,   is   recruited   to   DSB   by   poly-­‐[ADP-­‐ribose]  

(PAR)  (Polo  et  al.  (2010)  Embo  J;  Chou  et  al.  (2010)  Proc   Natl   Acad   Sci   USA).   CHD4   is   then   phosphorylated   by   the   Ataxia   telangiectasia   mutated   kinase   (ATM)   (Urquhart  et   al.   (2011)   Genome   Integr;   Chou  et   al.   (2010)   Proc   Natl   Acad   Sci   USA).   Consequently,   the   phosphorylated   CHD4   has   a   longer   residency   at   the   DSB.   It   has   been   suggested   that   NuRD   interferes   with   the   RNF8/RNF168   activity   (Larsen  et  al.  (2010)  J  Cell  Biol;  Smeenk  et  al.  (2010)  J  Cell   Biol).   The   loss   of   NuRD   subunits   increases   the   cellular   sensitivity   to   ionizing   radiation   or   ultraviolet   exposure   and   causes   spontaneous   DNA   breaks   (Smeenk   et   al.  

(2010)  J  Cell  Biol;  Li  et  al.  (2010)  J  Biol  Chem;  Larsen  et  al.  

(2010)   J   Cell   Biol).   Moreover,   it   has   been   suggested   that   CHD4   suppresses   the   transcription   by   inhibiting   the   formation  of  nascent  RNAs  and  the  elongating  Pol  II  (Chou   et   al.   (2010)   Proc   Natl   Acad   Sci   USA).   Finally,   during   the   DDR,   CHD4   regulates   the   transitions   of   G1/S   and   G2/M   (Smeenk  et  al.  (2010)  J  Cell  Biol;  Larsen  et  al.  (2010)  J  Cell   Biol;   Polo  et   al.   (2010)   Embo   J).   Unfortunately,   although   the  numerous  progress  made  to  understand  the  role  of  Mi-­‐

2  during  the  DDR,  its  exact  function  at  DSB  still  has  to  be   investigated.  

2.5.2.4  Higher  chromatin  structure    

Drosophila   has   the   particularity   that,   during   its   development,   DNA   undergoes   multiple   rounds   of   DNA   replication   without   performing   cytokinesis   in   some   tissues   (See   section   2.6.1).   It   creates   polytene   chromosomes,  where  hundreds  of  sister  chromatids  align.  

Those   chromosomes   share   similarities   with   interphase   chromosomes  of  diploid  cells.  

 It   has   been   suggested   that   the   chromatin   remodeler  ISWI  would  be  involved  in  the  regulation  of  the   polytene   chromosomes   structure   (Deuring  et   al.   (2000)   Mol  Cell).  More  recently,  the  implication  of  dMi-­‐2  has  also   been  highlighted  (Fasulo  et  al.  (2012)  PLoS  Genetics).  The   authors   showed   that   dMi-­‐2   facilitates   the   removal   of   cohesin  from  polytene  chromosomes  in  an  ATPase  activity   dependent   manner.   They   also   demonstrated   that   the   regulation  of  the  polytene  chromosomes  structure  is  not  a   common  function  among  the  CHD  chromatin  remodelers.  

Indeed,  an  over-­‐expression  of  Kismet,  a  homolog  of  CHD7,   does  not  influence  the  polytene  chromosomes  structure.    

At  a  different  structural  order,  it  has  been  shown   that  the  NuRD  complex  is  recruited  by  the  protein  hSATB1   (special   AT-­‐rich   sequence   binding   1)   to   repress   specific   loci,  in  human  cells  (Yasui  et  al.  (2002)  Nature).  hSATB1  is   a   nuclear   architectural   protein   that   regulates   gene   by   folding  chromatin  into  loops.  A  homolog  of  Mi-­‐2,  CHD8,  co-­‐

localizes   often   with   the   insulator-­‐binding   protein   hCTCF   (Ishihara  et  al.  (2006)  Mol  Cell).  The  interaction  of  CHD8-­‐

hCTCF   prevents   the   interplay   between   the   H19/IGF2   promoter   and   its   associated   enhancer.   However,   the   implications  of  Mi-­‐2  in  higher-­‐chromatin  structure  are  still   poorly   understood   and   additional   investigations   are   needed.  

 2.5.2.6   Development,   differentiation   and   lineage   commitment  

Based   on   several   studies   in   mammalian   and   in   Xenopus,  there  are  many  findings  reporting  that  the  NuRD   complex  is  involved  in  developmental  and  differentiation   processes   (Ahringer   (2000)   Trends   Genet;   Ramirez   and  

Hagman   (2009)   Epigenetics;   Reynolds   et   al.   (2013)   Development;   Marfella   and   Imbalzano   (2007)   Mutat   Res;  

Clapier   and   Cairns   (2009)   Annu   Rev   Biochem;   Ho   and   Crabtree  (2010)  Nature).  However,  the  Mec  functions  are   less   understood.     It   has   been   shown   by   the   group   of   Alexander  Brehm,  that  dMec  is  involved  in  the  repression   of  the  proneural  genes  of  the  acheate-­‐scute  locus  (Kunert   et   al.   (2009)   Embo   J).   Accordingly,   a   recent   study   by   the   group   of   Fritz   Müller,   in   C.   elegans,   describes   the   repression   of   the   proneural   genes   and   the   early   developmental   genes   by   Mec   (Passannante  et   al.   (2010)   PLoS  One).    

  The   developmental  Hox  genes   control   segmental   patterning.  Their  expression  is  regulated  by  two  opposing   groups   of   proteins:   the   Polycomb   group     (PcG)   and   the   Trithorax   group   (TrxG).   Kehle  et   al.   (1998)   showed   that   dMi-­‐2  is  required  to  repress  Hox  genes  (Kehle  et  al.  (1998)   Science).  Its  role  would  be  to  transit  the  initial  repression   signal   to   the   PcG   proteins.   In   other   models,   mCHD4   knocked   out   mice   are   viable   but   have   defects   in   their   hematopoietic  and  immune  systems  (Yoshida  et  al.  (2008)   Genes  Dev).  Together,  these  results  suggest  a  role  for  Mi-­‐2,   via  the  NuRD  or  the  Mec  complex,  in  the  development  of   several  species.  

CHD   remodelers   can   also   be   involved   in   cell   differentiation   and   cell   renewal.   By   example,   in   lymphocytes   B,   the   NuRD   complex   contains   the   subunit   MTA3   that   interacts   with   BCL-­‐6   and   blocks   the   B   cells   differentiation   (Fujita  et   al.   (2004)   Cell).   It   has   also   been   suggested   by   the   laboratory   of   James   Hagman   that   the   NuRD   complex   antagonizes   the   SWI/SNF-­‐mediated   gene   activation   of  ml-­1   and   blocks   the   maturation   of   B   cells   (Gao  et   al.   (2009)   Proc   Natl   Acad   Sci   USA).   In   a   different   cell   type,   the   thymocytes,   Mi-­‐2β   recruits   cofactors,   like   HEB  and  p300,  to  the  cd4  enhancer  (Williams  et  al.  (2004)  

Immunity).   Consequently,   in   the   vicinity   of   Mi-­‐2   binding   sites,  histones  are  hyper-­‐acetylated  and  the  cd4  enhancer   can  activate  gene  transcription.    

The   NuRD   complex   is   also   involved   in   lineage   commitment.   To   improve   our   understanding   of   the   Mi-­‐2   implication   in   lineage   commitment   processes,   several   studies   have   been   performed   in   mice.   In   murine   hematopoietic   stem   cells   (HSC),   Mi-­‐2/NuRD   acts   as   a   gatekeeper  for  the  cell  fate  determination  in  bone  marrow   (Yoshida   et   al.   (2008)   Genes   Dev).   It   inhibits   the   expression   of   genes   programmed   for   the   following   differentiation   steps   and   it   facilitates   the   expression   of   HSC-­‐specific   genes   (Zhang  et   al.   (2012)   Nat   Immunol).  

Additionally,   Mi-­‐2β   associates   with   Ikaros,   a   zinc   finger   DNA-­‐binding   protein   essential   for   lineage   determination   in   the   hematopoietic   cells,   to   inhibit   the  cd4   silencer   (Zhang   et   al.   (2012)   Nat   Immunol).   Additional   investigations   performed   with   different   animal   models   corroborate   a   Mi-­‐2   function   in   cell   lineage   commitment.  

For   instance,   the   two   Mi-­‐2   homologs   of  C.   elegans   have   been   shown   to   be   involved   in   the   Ras   signalling   cascade   leading   to   cell   fate   determination   during   the   hermaphrodite   development   (von   Zelewsky  et   al.   (2000)   Development;   Guerry  et   al.   (2007)   Dev   Biol).   Moreover,   Pickle,   the   Arabidopsis   Mi-­‐2   ortholog,   is   a   component   of   an  auxin-­‐signalling  pathway  that  is  involved  in  lateral  root   formation  (Fukaki  et  al.  (2006)  Plant  J).  Collectively,  these   findings   suggest   an   important   role   for   Mi-­‐2   in   development,  differentiation  and  lineage  commitment  and   thus,   independently   of   the   organism.   Either   the   NuRD   or   the   Mec   complex   could   mediate   the   Mi-­‐2   functions.  

However,   more   investigations   are   required   to   fully   understand  the  implications  of  Mi-­‐2  and  its  complexes  in   those  biological  processes.  

2.5.2.7  Pathologies  and  cancers