Nuclear compartmentalization and chromatin regulation An example of a functional cell biology analysis Data from: Bantignies, F., Grimaud, C., Lavrov, S., Gabut, M., and Cavalli, G. (2003). Inheritance of Polycomb-dependent chromosomal interactions in Drosophila. Genes Dev 17, 2406-2420. Grimaud, C., Bantignies, F., Pal-Bhadra, M., Ghana, P., Bhadra, U., and Cavalli, G. (2006). RNAi Components Are Required for Nuclear Clustering of Polycomb Group Response Elements. Cell 124, 957-971

PgG proteins form nuclear compartments PC protein has a speckled distribution in the cell nucleus Z axis image projections PC / Heterochromatin Different target genes may cluster at PcG foci. These associations may play a role in regulation of PcG targets

Chromosome pairing and PRE function During embryonic development, chromosome homologs pair in diptera. Pairing brings homolog sequences in close physical proximity. This pairing correlates with the strength of PcG and trxG mediated regulation Weak PcG mediated repression Strong PcG mediated repression PRE Heterozygous PRE Homozygous

An example of Pairing Sensitive silencing: the Fab-X transgenic line mini-white P scalloped (sd) Chromosome X Transgenic Fab-7 heterozygous Transgenic Fab-7 homozygous w w w ---> weak silencing of the mini-white reporter gene ---> strong mini-white silencing

In the line Fab-X, the Fab-7 transgenic PRE at the homozygous state represses an endogenous gene located close to the insertion site Fab-7 P mini-white P scalloped (sd) Chr. X 18 kb Heterozygous transgenic Fab-7 Homozygous transgenic Fab-7 sd sd sd sd No silencing of the endogenous sd gene Strong silencing of the endogenous sd gene

The sd repression mediated by the Fab-7 transgene requires the presence of the endogenous copy of Fab-7 Fab-X Fab-X; Fab71 sd sd Fab-7 transgene X X BX-C BX-C Endogenous Fab-7 III III Endogenous Fab-7 deletion, named Fab-71 Strong sd silencing dependent both on transgenic and endogenous Fab-7 sd derepression when the endogenous Fab-7 element is deleted

Do PRE lead to long-distance pairing? Three dimensional 2-color FISH in whole mount embryos 3D-Acquisition Fixation Permeabilisation Interphase nuclei Denaturation Hybridization Detection DAPI counterstating Dapi 5 mm BX-C locus Sd gene

PRE associate in the nucleus through DNA sequence homology Dapi Sd BX-C Merge WT Percentage of pairing sd - Fab-7 25 BX-C - 20 Fab-X sd Fab-7 - 15 Fab-7 BX-C - 10 Fab-X; Fab-71 - Fab-7 5 sd - BX-C WT Fab-X Fab-X; Fab-71 3mm Transgenic Fab-7 at sd (Chr.X) - + + Endogenous Fab-7 at the BX-C (Chr.III) + + -

Chromatin states can be inherited through meiosis by the following generations

Chromosome pairing and meiotic inheritance Genetic approach: 1. Erase trans homology by genetic crosses 2. Restore trans homology Strong PcG mediated repression Weak PcG mediated repression Pairing No pairing When pairing is disrupted, silencing is weakened. If pairing is re-established, can silencing be immediately re-acquired, or is the derepressed state inherited?

Meiotic inheritance of sd derepression Fab-X Fab-X Cross with Back cross with Fab-X; Fab71/+ Fab-X; Fab71 Fab-X F2 and F3 derepressed (20-25% sd) F0 repressed (85-90% sd) F1 partially derepressed (40-45% sd) Once established, the derepressed state can be inherited into a fraction of the progeny Is it because pairing can not be immediately re-established?

YES! Meiotic inheritance of sd derepression correlates with loss of Fab-7 pairing Dapi Sd BX-C Merge - 25 Fab-X - 20 - 15 % of pairing Fab-X after meiosis - 10 - 5 - Fab-X Fab-X after Meiosis

Nuclear architecture is heritable Multiple copies of 3.6 Kb of homologous DNA can find each other among 180Mb of genomic chromatin Chromatin pairing induces silencing and it is heritable Regulation of endogenous target genes of the Pc-G and of the trx-G may involve a precise compartmentalization in the cell nucleus. Nuclear compartmentalization is a heritable feature! Heterochromatin Target genes PcG bodies

Polycomb, nuclear organization and RNAi Manika Pal-Bhadra - IICT Charlotte Grimaud Frédéric Bantignies Collaborators: Utpal Bhadra - CCMB Manika Pal-Bhadra - IICT Hyderabad, India

RNAi and regulation of gene expression The RNAi machinery can induce post-transcriptional gene silencing by processing dsRNAs into short interfering RNAs of 21-23 nt that pair with the target mRNA and induce its degradation. The RNAi machinery is also responsible for the metabolism of endogenous noncoding transcripts, leading to the production of miRNAs. miRNAs can induce processing of mRNAs or block their transcription PTGS RNAi is also involved in transcriptional silencing in several species, like plants, Drosophila and S.pombe. In Drosophila, PcG components and RNAi members are required for a gene silencing process called cosuppression. We thus tested whether RNAi mutations affect the function of PREs TGS

Fab-7 dependent silencing depends on RNAi components Fab-X Fab-X;AGO1 45/72 Fab-X;dcr-2 L811fsX Fab-X;piwi 1 Fab-X;piwi 2 Fab-X;hls E1

PcG proteins form nuclear compartments called PcG bodies Z axis image projections PC / Heterochromatin Do RNAi component colocalize with PcG bodies?

Specific RNAi components colocalize with PcG bodies proteins DAPI PH merge Colocalization AGO1 42% Dcr-2 62% PIWI 52% Hls 19% Dcr-1 Very low

RNAi components are required for the recruitment of heterochromatin components to centromeres in S. pombe Is PcG protein recruitment dependent on RNAi components in Drosophila?

PcG proteins can be recruited to Fab-7 in most RNAi mutants DAPI FISH PC merge Fab-X Fab-X;AGO1 KO8121/45 The piwi gene is an exception ! Fab-X;piwi 1 Fab-X ; dcr-2 L811fsX

PcG protein targeting to Fab-7 in RNAi mutants DAPI sd BX-C PC merge w1118 Fab-X Fab-X Fab-X;dcr-2 L811fsX Loss of targeting only in piwi mutants Fab-X;piwi 2

…but not for targeting of PcG proteins at PREs Nuclear RNAi components are required for PcG mediated silencing of transgenes …but not for targeting of PcG proteins at PREs Are long-distance interactions of PREs affected in RNAi mutants?

Defective maintenance of Fab-7 long-distance interactions in RNAi mutant larvae Endogenous Fab-7 Transgenic Fab-7 DAPI sd BX-C merge w1118 + - Fab-X + + Fab-X;dcr-2 L811fsX + + Fab-X;piwi 2 + +

Summary RNAi components affect PcG-mediated gene silencing at the Fab-7 element They are not absolutely required for PcG recruitment at this PRE or at endogenous genes Most RNAi components are partly located in the nucleus, where they partially colocalize with PcG proteins The Fab-7 element can establish long-distance interactions that require a novel nuclear function of RNAi components We predict a general role of long-range associations and of the RNAi machinery in cosuppression/paramution phenomena