Concentrating factors in nuclear bodies is thought to promote efficient gene expression. Tatomer et al. show that the histone locus body (HLB) concentrates pre-mRNA processing factors at replication-dependent histone genes, resulting in optimal 3′ end formation of histone mRNAs coupled with transcription termination.

The Drosophila Multi Sex Combs (Mxc) protein is necessary for the recruitment of histone mRNA biosynthetic factors to the histone locus body (HLB). Mxc contains multiple domains required for HLB assembly and histone mRNA biosynthesis. Two N-terminal domains of Mxc are essential for promoting HLB assembly via a self-interaction.

Loss of the short arm of chromosome 3 (3p) occurs early in >95% of clear cell renal cell carcinoma (ccRCC). Nearly ubiquitous 3p loss in ccRCC suggests haploinsufficiency for 3p tumor suppressors as early drivers of tumorigenesis. We previously reported methyltransferase , which trimethylates H3 histones on lysine 36 (H3K36me3) and is located in the 3p deletion, to also trimethylate microtubules on lysine 40 (αTubK40me3) during mitosis, with αTubK40me3 required for genomic stability. We now show that monoallelic,-deficient cells retaining H3K36me3, but not αTubK40me3, exhibit a dramatic increase in mitotic defects and micronuclei count, with increased viability compared with biallelic loss. In -inactivated human kidney cells, rescue with a pathogenic mutant deficient for microtubule (αTubK40me3), but not histone (H3K36me3) methylation, replicated this phenotype. Genomic instability (micronuclei) was also a hallmark of patient-derived cells from ccRCC. These data show that the tumor suppressor displays a haploinsufficiency phenotype disproportionately impacting microtubule methylation and serves as an early driver of genomic instability. Loss of a single allele of a chromatin modifier plays a role in promoting oncogenesis, underscoring the growing relevance of tumor suppressor haploinsufficiency in tumorigenesis. .

Defects in apoptosis are not only a hallmark of cancer initiation and progression but can also underlie the development of chemoresistance. How the tightly regulated cascade of protein-protein interactions between members of three competing protein families regulating the apoptotic cascade is subverted in tumor cells is incompletely understood. Here, we show that KLF6-SV1, whose overexpression is associated with poor survival in several different cancers and is an alternatively spliced isoform of the Krüppel-like tumor suppressor KLF6, is a critical prosurvival/antiapoptotic protein. KLF6-SV1 binds the proapoptotic BH3-only protein NOXA, which results in their mutual HDM2-dependent degradation. In turn, this increases the intracellular concentration of the prosurvival binding partner of NOXA, Mcl-1, and effectively blocks apoptosis. In an ovarian cancer model, systemically delivered small interfering RNA against KLF6-SV1 induces spontaneous apoptosis of tumor cells, decreases tumor burden, and restores cisplatin sensitivity in vivo. Moreover, i.p. delivery of siKLF6-SV1 RNA halts ovarian tumor progression and improves median and overall survival (progression-free for >15 months; P < 0.0002) in mice in a dose-dependent manner. Thus, KLF6-SV1 represents a novel regulator of protein interactions in the apoptotic cascade and a therapeutically targetable control point.

Kruppel-like factor 6 splice variant 1 (KLF6-SV1) is an oncogenic splice variant of the KLF6 tumor suppressor gene that is specifically overexpressed in a number of human cancers. Previously, we have demonstrated that increased expression of KLF6-SV1 is associated with decreased survival in lung adenocarcinoma patient samples and that targeted reduction of KLF6-SV1 using siRNA induced apoptosis both alone and in combination with the chemotherapeutic drug cisplatin. Here, we demonstrate that chemoresistant lung cancer cells express increased levels of KLF6-SV1. Furthermore, targeted reduction of KLF6-SV1 using RNA interference restores chemotherapy sensitivity to lung cancer cells both in culture and in vivo through induction of apoptosis. Conversely, overexpression of KLF6-SV1 resulted in a marked reduction in chemotherapy sensitivity in a tumor xenograft model. Combined, these findings highlight a functional role for the KLF6-SV1 splice variant in the regulation of chemotherapy response in lung cancer and could provide novel insight into lung cancer therapy.

Diaphyseal medullary stenosis with malignant fibrous histiocytoma (DMS-MFH) is an autosomal-dominant syndrome characterized by bone dysplasia, myopathy, and bone cancer. We previously mapped the DMS-MFH tumor-suppressing-gene locus to chromosomal region 9p21-22 but failed to identify mutations in known genes in this region. We now demonstrate that DMS-MFH results from mutations in the most proximal of three previously uncharacterized terminal exons of the gene encoding methylthioadenosine phosphorylase, MTAP. Intriguingly, two of these MTAP exons arose from early and independent retroviral-integration events in primate genomes at least 40 million years ago, and since then, their genomic integration has gained a functional role. MTAP is a ubiquitously expressed homotrimeric-subunit enzyme critical to polyamine metabolism and adenine and methionine salvage pathways and was believed to be encoded as a single transcript from the eight previously described exons. Six distinct retroviral-sequence-containing MTAP isoforms, each of which can physically interact with archetype MTAP, have been identified. The disease-causing mutations occur within one of these retroviral-derived exons and result in exon skipping and dysregulated alternative splicing of all MTAP isoforms. Our results identify a gene involved in the development of bone sarcoma, provide evidence of the primate-specific evolution of certain parts of an existing gene, and demonstrate that mutations in parts of this gene can result in human disease despite its relatively recent origin.

Many membrane-less organelles form through liquid-liquid phase separation, but how their size is controlled and whether size is linked to function remain poorly understood. The Histone Locus Body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of histone mRNAs. Here, we show that Drosophila HLBs form through phase separation of the scaffold protein multi-sex combs (Mxc). The size of HLBs is controlled in a precise and dynamic manner that is dependent on the cell cycle and zygotic gene activation. Control of HLB growth is achieved by a mechanism integrating nascent mRNAs at the histone locus, which catalyzes phase separation, and the nuclear concentration of Mxc, which is controlled by the activity of cyclin-dependent kinases. Reduced Cdk2 activity results in smaller HLBs and the appearance of nascent, misprocessed histone mRNAs. Our experiments thus identify a mechanism linking nuclear body growth and size with gene expression.

Posttranslational modifications (PTMs) on microtubules differentiate these cytoskeletal elements for a variety of cellular functions. We recently identified SETD2 as a dual-function histone and microtubule methyltransferase, and methylation as a new microtubule PTM that occurs on lysine 40 of a-tubulin, which is trimethylated (a-TubK40me3) by SETD2. In the course of these studies, we generated polyclonal (a-TubK40me3 pAb) and monoclonal (a-TubK40me3 mAb) antibodies to a methylated a-tubulin peptide (GQMPSD-Kme3-TIGGGDC). Here, we characterize these antibodies, and the specific mono-, di-or trimethylated lysine residues they recognize. While both the pAb and mAb antibodies recognized lysines methylated by SETD2 on microtubules and histones, the clone 18 mAb was more specific for methylated microtubules, with little cross-reactivity for methylated histones. The clone 18 mAb recognized specific subsets of microtubules during mitosis and cytokinesis, and lacked the chromatin staining seen by immunocytochemistry with the pAb. Western blot analysis using these antibodies revealed that methylated a-tubulin migrated faster than unmethylated a-tubulin, suggesting methylation may be a signal for additional processing of a-tubulin and/or microtubules. As the first reagents that specifically recognize methylated a-tubulin, these antibodies are a valuable tool for studying this new modification of the cytoskeleton, and the function of methylated microtubules.