The essential Drosophila CLAMP protein differentially regulates non-coding roX RNAs in male and females

Abstract

Heterogametic species require chromosome-wide gene regulation to compensate for differences in sex chromosome gene dosage. In Drosophila melanogaster, transcriptional output from the single male X-chromosome is equalized to that of XX females by recruitment of the Male Specific Lethal (MSL) complex, which increases transcript levels of active genes two-fold. MSL complex contains several protein components and two non-coding roX (RNA on the X) RNAs that are transcriptionally activated by MSL complex. We previously discovered that targeting of MSL complex to the X-chromosome is dependent on the Chromatin-Linked Adapter for MSL Protein (CLAMP) zinc finger protein. To better understand CLAMP function, we used the CRISPR/Cas9 genome editing system to generate a frameshift mutation in the clamp gene that eliminates expression of CLAMP protein. We found that clamp null females die at the third instar larval stage, while almost all clamp null males die at earlier developmental stages. Moreover, we found that in clamp null females roX gene expression is activated whereas in clamp null males roX gene expression is reduced. Therefore, CLAMP regulates roX abundance in a sex-specific manner. Our results provide new insights into sex-specific gene regulation by an essential transcription factor.

Histone locus regulation by the Drosophila dosage compensation adaptor protein CLAMP

“…However, consistent with our current observations, later in vivo studies suggested that GAF is not present at the histone locus (O'Brien et al 1995;Bhat et al 1996). Our results suggest that the 61-kDa CLAMP protein (Urban et al 2017) is the likely ∼66-kDa protein bound to the H3-H4p in vivo that was identified >25 years ago (Gilmour et al 1989;Weber and Gilmour 1995).…”

“…5H). Because zygotic CLAMP protein is not produced in clamp 2 homozygous animals (Urban et al 2017), we conclude that maternally deposited CLAMP from clamp 2 heterozygous mothers persists at the histone locus throughout development of the larval salivary gland. Furthermore, the strong MTD does not deplete all maternally deposited clamp transcript or protein, and therefore a small amount of CLAMP remains at embryonic HLBs.…”

“…embryos in which clamp is depleted by RNAi. While almost all embryos laid by mothers with RNAi-reduced clamp levels are inviable, ∼30% of zygotically mutant animals homozygous for the clamp 2 -null allele survive until the third instar larval stage (Urban et al 2017). These data indicate that survival of clamp 2 larvae is likely due to maternal deposition of wild-type clamp mRNA or protein.…”

“…We reported previously that clamp 2 -null animals produce undetectable amounts of CLAMP protein by both Western blot of salivary gland tissue and polytene chromosome immunostaining (Urban et al 2017). However, our previously characterized affinity-purified anti-CLAMP antibody detected many cross-reacting proteins by Western blot (Fig.…”

DrosophilaHistone Locus Body assembly and function involves multiple interactions

“…Immunoprecipitation of chromatin preparations was performed using rabbit anti-CLAMP antibody (Rieder et al ., 2017) and a polyclonal rabbit IgG (Millipore-Sigma, 12-370). qPCR was performed as described in (Urban et al ., 2017). Three biological replicates and two technical replicates were used for each genotype.…”