Linking a lincRNA to active chromatin

Wang et al show that a lincRNA encoded at one end of the HOXA gene cluster acts as a transcriptional enhancer, necessary for the translation of high order chromosomal structure into a transcriptionally active chromatin state.

Hox genes encode transcription factors that determine positional identities along the anterior-posterior (a-p) body axis and along the proximal-distal (p-d) axes of appendages. In vertebrates, Hox genes are found in four clusters, in which their 3′-5′ genomic arrangement mirrors their a-p and p-d expression patterns. For instance, genes found at 3′ end of the HOXA cluster such as HOXA1 and HOXA2 are necessary for specifying positional identities in hindbrain, whilst genes found at the 5′ end of the cluster such as HOXA13 and HOXA11 determine distal elements of the limbs.

To investigate how this genomic colinearity is translated into differential p-d expression patterns, Wang et al analysed chromosomal conformation at the HOXA locus in different human fibroblast cells. In distally derived cells (foreskin or foot fibroblasts) the 5′ end of the HOXA cluster displayed a compact and looped conformation, whilst the 3′ end seemed largely linear. An opposite conformation was found in proximally derived cells (lung fibroblasts). These findings correlated with the presence of specific histone post-translational modifications (PTMs). Areas of high chromatin interactions showed high levels of trimethylated histone H3 lysine 4 (H3K4me3) (associated with transcriptionally active chromatin), and low levels of histone H3 lysine 27 trimethylation (H3K27me3) (associated with transcriptionally silent chromatin).

histone lysine methylation states across the HOXA cluster compared between distal and proximal cells.

A lincRNA named HOTTIP is encoded at the 5′ end of the HOXA locus. Analysis of it’s expression revealed that it is transcribed in posterior and distal territories, in a manner similar to nearby HOXA genes. When HOTTIP RNA was depleted (using small interfering RNAs) in distal cells, expression of 5′ HOXA genes was abrogated. HOXA genes nearest the HOTTIP gene were the most effected, however transcriptional activity over 40kb of the HOXA locus was lessened. HOTTIP RNA depletion did not effect the expression of other genes tested, such as the highly homologous HOXD genes.

HOTTIP RNA depleted distal cells did not show any changes to the higher order chromosomal conformation at the HOXA locus, however they did display a broad loss of H3K4me3 at the 5′ end of the cluster. This was not mirrored by a concomitant gain of H3K27me3. Therefore it appears the loss of 5′ HOXA gene expression upon HOTTIP RNA depletion is linked to the loss of H3K4me3.

Effects of HOTTIP RNA depletion on histone lysine methylation states.

H3K4 methylation and it’s maintenance are mediated by protein complexes composed of lysine methyltransferases such as MLL1 and associated proteins like WDR5. Analysis of MLL1 and WDR5 occupancy in the HOXA cluster of distal fibroblasts revealed occupancy peaks at the transcriptional start sites of the 5′ located genes. These occupancy peaks disappeared when HOTTIP RNA was knocked down.Wang et al go on to show that HOTTIP RNA physically interacts with WDR5 protein.

Effects of HOTTIP RNA depletion on occupancy of MLL1 and WRD5.

A number of lines of evidence suggest that HOTTIP RNA acts in cis to regulate 5′ HOXA genes. For instance, HOTTIP RNA is expressed at very low copy number, and depletion did not have any effect on the HOXD locus. Retroviral insertion driven overexpression of HOTTIP RNA did not ectopically activate HOXA genes, nor could it rescue depletion of the endogenous HOTTIP RNA.

Tying these results together yields a model in which chromosomal looping brings HOTTIP RNA (specifically bound to the HOTTIP gene) into contact with the 5′ HOXA genes. HOTTIP lincRNA binds and targets WDR5/MLL complexes to the 5′ HOXA locus, creating a domain of H3K4me3 and transcriptional activation.

lincRNAs are probably a heterogeneous class of molecules, as they are arbitrarily defined on the basis of length. These results however, suggest what could be quite a common mechanism for how lincRNAs implicated in enhancer function can effect gene activation and program chromatin states.

See also: lincRNAs in development and evolution

Wang, K., Yang, Y., Liu, B., Sanyal, A., Corces-Zimmerman, R., Chen, Y., Lajoie, B., Protacio, A., Flynn, R., Gupta, R., Wysocka, J., Lei, M., Dekker, J., Helms, J., & Chang, H. (2011). A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression Nature, 472 (7341), 120-124 DOI: 10.1038/nature09819

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