Dosage compensation is a fundamental mechanism in many species that corrects for the inherent imbalance in X-chromosome copy number between XY males and XX females. 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 to specific sequences along the length of the X-chromosome. The initial recruitment of MSL complex to the X-chromosome is dependent on a recently discovered zinc finger protein called Chromatin-Linked Adapter for MSL Proteins (CLAMP). However, further studies on the in vivo function of CLAMP remained difficult because the location of the gene in pericentric heterochromatin made it challenging to create null mutations or deficiencies. Using the CRISPR/Cas9 genome editing system, we generated the first null mutant in the clamp gene that eliminates expression of CLAMP protein. We show that CLAMP is necessary for both male and female viability. While females die at the third instar larval stage, males die earlier, likely due to the essential role of CLAMP in male dosage compensation. Moreover, we demonstrate that CLAMP promotes dosage compensation in males and represses key male-specific transcripts involved in sex-determination in females. Our results reveal that CLAMP is an essential protein with dual roles in males and females, which together assure that dosage compensation is a sex-specific process.

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 2-fold. The MSL complex contains several protein components and two non-coding RNA on the X ( roX) RNAs that are transcriptionally activated by the MSL complex. We previously discovered that targeting of the MSL complex to the X-chromosome is dependent on the chromatin-linked adapter for MSL proteins (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 the 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.

Dosage compensation adjusts the expression levels of genes on one or both targeted sex chromosomes in heterogametic species. This process results in the normalized transcriptional output of important and essential gene families encoded on multiple chromosomes. The mechanisms of dosage compensation have been studied in many model organisms, including Drosophila melanogaster (fly), Caenorhabditis elegans (worm), and Mus musculus (mouse). Although the mechanisms of dosage compensations differ among these species, all of these processes rely on the initial discrimination of the X chromosome from autosomes. Recently, a new paradigm for how the X chromosome is targeted for regulation was identified in Drosophila. This mechanism involves a newly identified zinc finger protein, CLAMP. Here, we review important factors involved in dosage compensation across species with special focus on the fly. Understanding how the newly identified CLAMP protein is involved in X targeting in the fly could provide key insights into how the X chromosome is initially identified across species.

The Drosophila male-specific lethal (MSL) dosage compensation complex increases transcript levels on the single male X chromosome to equal the transcript levels in XX females. However, it is not known how the MSL complex is linked to its DNA recognition elements, the critical first step in dosage compensation. Here, we demonstrate that a previously uncharacterized zinc finger protein, CLAMP (chromatin-linked adaptor for MSL proteins), functions as the first link between the MSL complex and the X chromosome. CLAMP directly binds to the MSL complex DNA recognition elements and is required for the recruitment of the MSL complex. The discovery of CLAMP identifies a key factor required for the chromosome-specific targeting of dosage compensation, providing new insights into how subnuclear domains of coordinate gene regulation are formed within metazoan genomes.

Sex chromosome dosage compensation in Drosophila provides a model for understanding how chromatin organization can modulate coordinate gene regulation. Male Drosophila increase the transcript levels of genes on the single male X approximately two-fold to equal the gene expression in females, which have two X-chromosomes. Dosage compensation is mediated by the Male-Specific Lethal (MSL) histone acetyltransferase complex. Five core components of the MSL complex were identified by genetic screens for genes that are specifically required for male viability and are dispensable for females. However, because dosage compensation must interface with the general transcriptional machinery, it is likely that identifying additional regulators that are not strictly male-specific will be key to understanding the process at a mechanistic level. Such regulators would not have been recovered from previous male-specific lethal screening strategies. Therefore, we have performed a cell culture-based, genome-wide RNAi screen to search for factors required for MSL targeting or function. Here we focus on the discovery of proteins that function to promote MSL complex recruitment to "chromatin entry sites," which are proposed to be the initial sites of MSL targeting. We find that components of the NSL (Non-specific lethal) complex, and a previously unstudied zinc-finger protein, facilitate MSL targeting and display a striking enrichment at MSL entry sites. Identification of these factors provides new insight into how MSL complex establishes the specialized hyperactive chromatin required for dosage compensation in Drosophila.