11 Hox Genes - University of Texas at Austinw3.biosci.utexas.edu/atkinson/Epigenetics2014...Hox...

Hox genesEstablish body plan during developmentSpecify head to tail axis of animal embryosHead Hox genes, abdomen hox genes.Mutations can cause one body part to transform to another39 transcription factors genes organized in 4 clustersFour clusters called HOXA to HOXDSpecify positional identity of cells

Hox genes

Discovered in Drosophila in 1923 by Bridges and Morgan

Antennapaedia complex ANT-CBithorax complex BX-C

Hox genes

Hox genes show colinearity

Colinearity - order of genes is same as the head-to-tail expression pattern!!!

Temporal and spatial expression pattern is correlated to location in cluster!

Mallo, M and Alonso, CR (2013). The regulation of Hox gene expression during animal development. Development 140:3951-3963.

Hox genes show colinearity

This expression is maintained epigenetically.

Mallo, M and Alonso, CR (2013). The regulation of Hox gene expression during animal development. Development 140:3951-3963.

This relationship is evolutionarily conserved

vertebrates had 2 rounds

of genome duplication

Lemons, D and McGinnis, W (2006). Genomic evolution of Hox gene clusters. Science 313:1918-1922.

This relationship is evolutionarily conserved

Expression is molded by transcription factors involved in earlier steps of developmenta memory system that uses Polycomb (PcG) and Trithorax (TrxG) group proteinsregulation of one Hox gene by anothermicroRNAslncRNAs

This head to toe expression pattern is produced by regulation at many

levels during gene expression.

Epigenetic regulation

Transcriptional activation produced by transcription factors in early embryo are remembered through the action of Polycomb (PcG) and Trithorax (TrxG) group transcription factors.

Trithorax group (TrxG) H3K4 HMTaseproduces H3K4me3 which maintains open chromatinPolycomb group (PcG) - two complexes belong here PRC1 and PRC2

PRC2 - H3K27 HMTasemaintains closed chromatinPRC1 binds H3K27me3 and ubiquitylates H2A at K119

Montavon, T and Duboule, D (2013). Chromatin organization and global regulation of Hox gene clusters. Philos Trans R Soc Lond B Biol Sci 368:20120367.

Epigenetic regulation

Representation of different aspects of Hox gene regulation associated with chromatin/nuclear characteristics.

Mallo M , and Alonso C R Development 2013;140:3951-3963

Vertebrates

EmbryonicStem cells

As the embryo develops

H3K27me3 is removed and

H3K4me3 appears.

two epigenetic states, in a window that shifts along with time,from one extremity of the cluster to the other. The analysis ofembryos carrying an engineered split HoxD cluster furtherrevealed that clustering is not necessary for the initial depositionofH3K27me3, yet it is required for a fully coordinated transitionin histone modifications [48].

The mechanisms recruiting PcG proteins to their targetloci are not fully understood. In Drosophila, relatively shortsequences termed Polycomb response elements (PREs) seemto be both necessary and sufficient for PcG recruitment andgene silencing, although these sites cannot be defined by a con-sensus DNA sequence [25]. The situation is more complex invertebrates, where classical PREs—as defined in flies—remainto be identified. For instance, an element from the humanHOXD cluster was recently reported to bind PcG proteinsand to induce repression of a reporter gene [49]. However,the deletion of the murine orthologous sequence did notcause any dramatic change in Hoxd gene regulation [50],suggesting that it is not critical for PcG recruitment or,alternatively, that it is part of a robust mechanism with highcompensatory capacities. Interestingly, CG-rich sequences,which are particularly abundant within Hox clusters, were pro-posed as a hallmark of PcG target promoters, at least in ES cells[51,52]. Silencing at these particular loci might thus involve thecooperative activity of multiple DNA elements.

4. Higher-order chromatin organizationThe coordinated control of these dynamic epigenetic statesmight be facilitated by the spatial compartmentalization of

Hox clusters. Changes in higher-order chromatin organizationof Hox clusters were first observed by microscopy approachessuch as fluorescent in situ hybridization (FISH), which allowthe visualization of specific loci within the nucleus. Differen-tiation of ES cells after retinoic acid treatment leads to adecompaction of the HoxB cluster that parallels its global tran-scriptional activation [53]. Furthermore, Hox clusters are alsoless compact in those embryonic territories where Hox genesare probablyactive, than in silent regions [54].However, the res-olution of this approach did not allow the stepwise, collineartransition to be documented.

Also, owing to its limited resolution, FISH cannot cur-rently provide a precise mapping of the three-dimensionalorganization of Hox loci, in either their active or inactivestates. The development of the chromosome conformationcapture (3C) technique, which provides an estimation ofthe average frequency of specific DNA–DNA ‘contacts’ (orproximity, see below) within a cell population, has greatlyhelped to overcome this limitation. While early 3C analysesonly addressed interactions between a limited number ofsequences, variant approaches such as 4C (circular 3C), 5C(3C carbon copy) or Hi-C generate rather unbiased datasets,where themapping of all sequences contacting a locus of inter-est, or the analysis of mutual interactions between a largenumber of pre-determined sites—or even within an entiregenome, can be produced [55]. Using these approaches,changes in the three-dimensional organization of Hox clusterswere detected upon cell differentiation in culture or betweencell lines derived from various regions of the body [56–58].

In dissected murine embryonic tissues, this configuration istightly associated with the transcriptional activity of the gene

K27K27K27K27K27K27K27K27K27K27

K4K4K4K4K4K4K27K27K27K27

K27 demethylation, K4 methylation


K4K4K4K4K4K4K4 K4K4K4

13 12 11 10 9 8 4 3 1(a)

(i)

(ii)

(iii)

(i)

(ii)

(iii)

anterior

posterior

silent

active

(b)

Figure 1. Collinearity during trunk extension and chromatin dynamics at Hox clusters. Expression of Hox gene along the anterior-to-posterior (AP) embryonic axis iscollinear with gene order within the cluster. (a) During axial extension, the sequential onset of Hox gene transcriptional activation is accompanied by a transition inhistone modifications over the gene cluster. In ES cells (i), the whole cluster is labelled with H3K27me3 (orange), a mark associated with Polycomb-mediatedsilencing. In the developing embryo, this mark is progressively erased and replaced by H3K4me3 (green), concomitantly with gene activation. (b) Active andsilent Hox loci segregate into distinct spatial compartments along the AP axis. In embryonic tissues where the whole cluster is repressed, such as the forebrain(i), Hox clusters form a compact three-dimensional structure. In regions where subsets of Hox genes are expressed (anterior trunk, ii), active and silent genessegregate in distinct compartments, labelled with either H3K27me3 (silent compartment) or H3K4me3 (active compartment). In posterior embryonic regions(iii), most genes are transcribed and participate in the active compartment.

rstb.royalsocietypublishing.orgPhilTransR

SocB368:20120367

3

on March 17, 2014rstb.royalsocietypublishing.orgDownloaded from


two epigenetic states, in a window that shifts along with time,from one extremity of the cluster to the other. The analysis ofembryos carrying an engineered split HoxD cluster furtherrevealed that clustering is not necessary for the initial depositionofH3K27me3, yet it is required for a fully coordinated transitionin histone modifications [48].

The mechanisms recruiting PcG proteins to their targetloci are not fully understood. In Drosophila, relatively shortsequences termed Polycomb response elements (PREs) seemto be both necessary and sufficient for PcG recruitment andgene silencing, although these sites cannot be defined by a con-sensus DNA sequence [25]. The situation is more complex invertebrates, where classical PREs—as defined in flies—remainto be identified. For instance, an element from the humanHOXD cluster was recently reported to bind PcG proteinsand to induce repression of a reporter gene [49]. However,the deletion of the murine orthologous sequence did notcause any dramatic change in Hoxd gene regulation [50],suggesting that it is not critical for PcG recruitment or,alternatively, that it is part of a robust mechanism with highcompensatory capacities. Interestingly, CG-rich sequences,which are particularly abundant within Hox clusters, were pro-posed as a hallmark of PcG target promoters, at least in ES cells[51,52]. Silencing at these particular loci might thus involve thecooperative activity of multiple DNA elements.

4. Higher-order chromatin organizationThe coordinated control of these dynamic epigenetic statesmight be facilitated by the spatial compartmentalization of

Hox clusters. Changes in higher-order chromatin organizationof Hox clusters were first observed by microscopy approachessuch as fluorescent in situ hybridization (FISH), which allowthe visualization of specific loci within the nucleus. Differen-tiation of ES cells after retinoic acid treatment leads to adecompaction of the HoxB cluster that parallels its global tran-scriptional activation [53]. Furthermore, Hox clusters are alsoless compact in those embryonic territories where Hox genesare probablyactive, than in silent regions [54].However, the res-olution of this approach did not allow the stepwise, collineartransition to be documented.

Also, owing to its limited resolution, FISH cannot cur-rently provide a precise mapping of the three-dimensionalorganization of Hox loci, in either their active or inactivestates. The development of the chromosome conformationcapture (3C) technique, which provides an estimation ofthe average frequency of specific DNA–DNA ‘contacts’ (orproximity, see below) within a cell population, has greatlyhelped to overcome this limitation. While early 3C analysesonly addressed interactions between a limited number ofsequences, variant approaches such as 4C (circular 3C), 5C(3C carbon copy) or Hi-C generate rather unbiased datasets,where themapping of all sequences contacting a locus of inter-est, or the analysis of mutual interactions between a largenumber of pre-determined sites—or even within an entiregenome, can be produced [55]. Using these approaches,changes in the three-dimensional organization of Hox clusterswere detected upon cell differentiation in culture or betweencell lines derived from various regions of the body [56–58].

In dissected murine embryonic tissues, this configuration istightly associated with the transcriptional activity of the gene

K27K27K27K27K27K27K27K27K27K27

K4K4K4K4K4K4K27K27K27K27



K4K4K4K4K4K4K4 K4K4K4

13 12 11 10 9 8 4 3 1(a)

(i)

(ii)

(iii)

(i)

(ii)

(iii)

anterior

posterior

silent

active

(b)

Figure 1. Collinearity during trunk extension and chromatin dynamics at Hox clusters. Expression of Hox gene along the anterior-to-posterior (AP) embryonic axis iscollinear with gene order within the cluster. (a) During axial extension, the sequential onset of Hox gene transcriptional activation is accompanied by a transition inhistone modifications over the gene cluster. In ES cells (i), the whole cluster is labelled with H3K27me3 (orange), a mark associated with Polycomb-mediatedsilencing. In the developing embryo, this mark is progressively erased and replaced by H3K4me3 (green), concomitantly with gene activation. (b) Active andsilent Hox loci segregate into distinct spatial compartments along the AP axis. In embryonic tissues where the whole cluster is repressed, such as the forebrain(i), Hox clusters form a compact three-dimensional structure. In regions where subsets of Hox genes are expressed (anterior trunk, ii), active and silent genessegregate in distinct compartments, labelled with either H3K27me3 (silent compartment) or H3K4me3 (active compartment). In posterior embryonic regions(iii), most genes are transcribed and participate in the active compartment.

rstb.royalsocietypublishing.orgPhilTransR

SocB368:20120367

3

on March 17, 2014rstb.royalsocietypublishing.orgDownloaded from

EmbryonicStem cells

As the embryo develops

H3K27me3 is removed and

H3K4me3 appears.


Repression promotes movement and movement promotes repression.

Vertebrates

Temporal lncRNA

transcribed from between HOXC11 and HOXC12

Blocks expression in posterior Hox D cluster

recruits PRC2 and the LSD histone demethylation

Frances R Goodman . Congenital abnormalities of body patterning: embryology revisited/ The Lancet, Volume 362, Issue 9384, Pages 651 - 662, 23 August 2003

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Fatica, A and Bozzoni, I (2013). Long non-coding RNAs: new players in cell differentiation and development. Nat Rev Genet

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