Using a combination of techniques (e4C, 3C, RNA FISH, DNA FISH and immuno-FISH), we have shown that the mouse globin genes preferentially associate with hundreds of other transcribed genomic loci in transcription factories. The globin-interacting genes are distributed over nearly all mouse chromosomes, uncovering extensive intra- and interchromosomal transcriptional interaction networks in erythroid nuclei. It is highly unlikely that all of these interactions occur simultaneously in the same cell. Instead, our data on gene triplet associations at factories imply that genome organization is inherently plastic. We propose that multiple different genome conformations exist and that each brings the globin genes into close proximity with a varied subset of their preferred transcriptional partners (). Although gene associations at factories seem to be dynamic44
, it is presently not known whether these whole-genome conformations are dynamically interchangeable within one nucleus or whether individual conformations are relatively stable in a subset of nuclei.
Within the globin transcriptional networks, Klf1-regulated genes are preferentially transcribed at a limited number of specialized transcription factories containing large amounts of Klf1. Our data also show that Klf1-regulated genes share Klf1-containing factories and that Klf1 is required for clustering of these co-regulated genes. It is important to point out that the majority of Klf1-regulated genes are not dependent on Klf1 for expression but instead require Klf1 for increased expression in the definitive erythroid lineage. For example, many of the genes encoding proteins involved in iron uptake and heme synthesis are ubiquitously expressed in all cell types but are highly upregulated by Klf1 during erythropoiesis36,37
. We propose that specialized transcription factories boost the expression of clustered, co-regulated genes by concentrating specific transcription factors required for their coordinate or increased transcription (). This may occur through a self-organization process whereby locally elevated concentrations of transcription factors and their cognate binding sites increase the probability of gene re- initiation, thus increasing occupancy time and transcriptional output at a shared factory. Such a model does not propose that specific pairwise interactions of genes are essential for their expression, but rather that individual genes may indirectly benefit from cooperative associations in these specialized microenvironments. Consequently, removal of a member of the network would not be expected to affect transcription of other network members to any great extent. In agreement with this, transcription and expression of several genes that associate with Hbb
did not change in Hbb
LCR knockout mice (data not shown), in which Hbb
factory association and expression are markedly reduced15
The concept of transcription factor–mediated spatial genome organization is supported by the observation that ectopic Klf1-regulated transgenes enter the same transcription sites as a co-regulated endogenous gene. This finding implies that transcriptional associations may considerably influence genome organization. This is supported indirectly by observed alterations in chromosomal co-associations when transcription is inhibited5,13
. Thus, we propose that preferential associations in transcription factories substantially affect higher order chromosomal conformations and are a major driving force in tissue-specific chromosome positioning45
Our results contrast with previous subgenomic 4C studies that detected few interchromosomal contacts for Hbb
and concluded that active Hbb
loci interact primarily in cis28
, regardless of transcription46
. We propose that the reasons for this discrepancy are probably the increased sensitivity and specificity of e4C. The biotin pre-enrichment step removes the thousands of copies of genomic DNA in the PCR reaction, which contribute to the probe in other 4C assays, potentially causing background noise upon microarray hybridization. In addition, the RNAPII-S5P ChIP step specifically enriches for transcriptionally active alleles, thus focusing on interactions at transcription factories and excluding cells not transcribing the bait gene. Conventional 3C and 4C approaches inherently average the interactions of all alleles in a population, active and temporarily nontranscribed; thus, preferential associations among the transcribed subpopulation of alleles might escape detection with these techniques.
Previous studies have focused on small numbers of genes or genetic elements that functionally cluster in three dimensions, such as nucleolar rDNA repeats47
, tRNA genes in yeast48
, silenced Hox
genes in Drosophila49
, virally induced associations between the interferon-β gene and NF-κB–bound sites6
, between EZH2-bound sites50
, and clustering of transiently transfected reporter constructs51
. Here we used a genome-wide e4C screen to identify extensive spatial networks of active genes, demonstrating that interchromosomal clustering of genes is a widespread principle of nuclear organization.
Our results imply that transcriptional regulation should be considered in the context of the three-dimensional organization of the genome rather than as a mechanism that acts on single genes in isolation. Co-regulated genes cluster in nuclear space and potentially collaborate to create transcription sites that are optimized for their regulated expression. Thus, the concept of regulation of cell-type gene expression patterns by combinatorial transcription factor control is played out in the nucleus as combinatorial associations between multiple genes at specialized transcription sites, creating functional overlapping transcription networks.