Shinpei Kawaoka

Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation

Cancer cells frequently depend on chromatin regulatory activities to maintain a malignant phenotype. Here, we show that leukemia cells require the mammalian SWI/SNF chromatin remodeling complex for their survival and aberrant self-renewal potential. While Brg1, an ATPase subunit of SWI/SNF, is known to suppress tumor formation in several cell types, we found that leukemia cells instead rely on Brg1 to support their oncogenic transcriptional program, which includes Myc as one of its key targets. To account for this context-specific function, we identify a cluster of lineage-specific enhancers located 1.7 Mb downstream from Myc that are occupied by SWI/SNF as well as the BET protein Brd4. Brg1 is required at these distal elements to maintain transcription factor occupancy and for long-range chromatin looping interactions with the Myc promoter. Notably, these distal Myc enhancers coincide with a region that is focally amplified in ∼3% of acute myeloid leukemias. Together, these findings define a leukemia maintenance function for SWI/SNF that is linked to enhancer-mediated gene regulation, providing general insights into how cancer cells exploit transcriptional coactivators to maintain oncogenic gene expression programs.

A single female-specific piRNA is the primary determiner of sex in the silkworm

The silkworm Bombyx mori uses a WZ sex determination system that is analogous to the one found in birds and some reptiles. In this system, males have two Z sex chromosomes, whereas females have Z and W sex chromosomes. The silkworm W chromosome has a dominant role in female determination, suggesting the existence of a dominant feminizing gene in this chromosome. However, the W chromosome is almost fully occupied by transposable element sequences, and no functional protein-coding gene has been identified so far. Female-enriched PIWI-interacting RNAs (piRNAs) are the only known transcripts that are produced from the sex-determining region of the W chromosome, but the function(s) of these piRNAs are unknown. Here we show that a W-chromosome-derived, female-specific piRNA is the feminizing factor of B. mori. This piRNA is produced from a piRNA precursor which we named Fem. Fem sequences were arranged in tandem in the sex-determining region of the W chromosome. Inhibition of Fem-derived piRNA-mediated signalling in female embryos led to the production of the male-specific splice variants of B. mori doublesex (Bmdsx), a gene which acts at the downstream end of the sex differentiation cascade. A target gene of Fem-derived piRNA was identified on the Z chromosome of B. mori. This gene, which we named Masc, encoded a CCCH-type zinc finger protein. We show that the silencing of Masc messenger RNA by Fem piRNA is required for the production of female-specific isoforms of Bmdsx in female embryos, and that Masc protein controls both dosage compensation and masculinization in male embryos. Our study characterizes a single small RNA that is responsible for primary sex determination in the WZ sex determination system.

Large Scale Full-Length cDNA Sequencing Reveals a Unique Genomic Landscape in a Lepidopteran Model Insect, Bombyx mori

The establishment of a complete genomic sequence of silkworm, the model species of Lepidoptera, laid a foundation for its functional genomics. A more complete annotation of the genome will benefit functional and comparative studies and accelerate extensive industrial applications for this insect. To realize these goals, we embarked upon a large-scale full-length cDNA collection from 21 full-length cDNA libraries derived from 14 tissues of the domesticated silkworm and performed full sequencing by primer walking for 11,104 full-length cDNAs. The large average intron size was 1904 bp, resulting from a high accumulation of transposons. Using gene models predicted by GLEAN and published mRNAs, we identified 16,823 gene loci on the silkworm genome assembly. Orthology analysis of 153 species, including 11 insects, revealed that among three Lepidoptera including Monarch and Heliconius butterflies, the 403 largest silkworm-specific genes were composed mainly of protective immunity, hormone-related, and characteristic structural proteins. Analysis of testis-/ovary-specific genes revealed distinctive features of sexual dimorphism, including depletion of ovary-specific genes on the Z chromosome in contrast to an enrichment of testis-specific genes. More than 40% of genes expressed in specific tissues mapped in tissue-specific chromosomal clusters. The newly obtained FL-cDNA sequences enabled us to annotate the genome of this lepidopteran model insect more accurately, enhancing genomic and functional studies of Lepidoptera and comparative analyses with other insect orders, and yielding new insights into the evolution and organization of lepidopteran-specific genes.

Histone H2B ubiquitin ligase RNF20 is required for MLL-rearranged leukemia

Mixed-lineage leukemia (MLL) fusions are potent oncogenes that initiate aggressive forms of acute leukemia. As aberrant transcriptional regulators, MLL-fusion proteins alter gene expression in hematopoietic cells through interactions with the histone H3 lysine 79 (H3K79) methyltransferase DOT1L. Notably, interference with MLL-fusion cofactors like DOT1L is an emerging therapeutic strategy in this disease. Here, we identify the histone H2B E3 ubiquitin ligase ring finger protein 20 (RNF20) as an additional chromatin regulator that is necessary for MLL-fusion–mediated leukemogenesis. Suppressing the expression of Rnf20 in diverse models of MLL-rearranged leukemia leads to inhibition of cell proliferation, under tissue culture conditions as well as in vivo. Rnf20 knockdown leads to reduced expression of MLL-fusion target genes, effects resembling Dot1l inhibition. Using ChIP-seq, we found that H2B ubiquitination is enriched in the body of MLL-fusion target genes, correlating with sites of H3K79 methylation and transcription elongation. Furthermore, Rnf20 is required to maintain local levels of H3K79 methylation by Dot1l at Hoxa9 and Meis1. These findings support a model whereby cotranscriptional recruitment of Rnf20 at MLL-fusion target genes leads to amplification of Dot1l-mediated H3K79 methylation, thereby rendering leukemia cells dependent on Rnf20 to maintain their oncogenic transcriptional program.

Developmentally synchronized expression of two Bombyx mori Piwi subfamily genes, SIWI and BmAGO3 in germ-line cells

The Piwi subfamily proteins and their partner, Piwi-interacting RNAs (piRNAs), play an important roles in germ-line development and silencing of selfish DNA elements. To date, however, the developmental expression profiles of the Piwi subfamily genes are poorly known. In this study, we examined the expression profiles of two Bombyx mori Piwi subfamily genes, silkworm Piwi (SIWI) and BmAGO3, which are possible partners of Bombyx piRNA-like small RNAs we recently identified in B. mori germ-line cells. Quantitative reverse transcription-polymerase chain reaction analyses demonstrated that these two genes were abundantly expressed in the larval testis, pupal ovary, and adult eggs, suggesting that they might be involved in spermatogenesis and oogenesis in Bombyx. Notably, developmental expression patterns of SIWI and BmAGO3 were remarkably similar. Collectively, our results suggest that SIWI and BmAGO3 may cooperate in an unknown pathway during the development of B. mori germ-line cells.

A role for transcription from a piRNA cluster in de novo piRNA production

PIWI-interacting RNAs (piRNAs) are at the heart of the nucleic acid-based adaptive immune system against transposons in animal gonads. To date, how the piRNA pathway senses an element as a substrate and how de novo piRNA production is initiated remain elusive. Here, by utilizing a GFP transgene, we screened and obtained clonal silkworm BmN4 cell lines producing massively amplified GFP-derived piRNAs capable of silencing GFP in trans. In multiple independent cell lines where GFP expression was silenced by the piRNA pathway, we detected a common transcript from an endogenous piRNA cluster, in which a part of the cluster is uniquely fused with an antisense GFP sequence. Bioinformatic analyses suggest that the fusion transcript is a source of GFP primary piRNAs. Our data implicate a role for transcription from a piRNA cluster in initiating de novo piRNA production against a new insertion.

The silkworm W chromosome is a source of female-enriched piRNAs

In the silkworm, Bombyx mori, the W chromosome plays a dominant role in female determination. However, neither protein-coding genes nor transcripts have so far been isolated from the W chromosome. Instead, a large amount of functional transposable elements and their remnants are accumulated on the W chromosome. PIWI-interacting RNAs (piRNAs) are 23-30-nt-long small RNAs that potentially act as sequence-specific guides for PIWI proteins to silence transposon activity in animal gonads. In this study, by comparing ovary- and testis-derived piRNAs, we identified numerous female-enriched piRNAs. Our data indicated that female-enriched piRNAs are derived from the W chromosome. Moreover, comparative analyses on piRNA profiles from a series of W chromosome mutant strains revealed a striking enrichment of a specific set of transposon-derived piRNAs in the putative sex-determining region. Collectively, we revealed the nature of the silkworm W chromosome as a source of piRNAs.

Zygotic amplification of secondary piRNAs during silkworm embryogenesis

PIWI-interacting RNAs (piRNAs) are 23-30-nucleotide-long small RNAs that act as sequence-specific silencers of transposable elements in animal gonads. In flies, genetics and deep sequencing data have led to a hypothesis for piRNA biogenesis called the ping-pong cycle, where antisense primary piRNAs initiate an amplification loop to generate sense secondary piRNAs. However, to date, the process of the ping-pong cycle has never been monitored at work. Here, by large-scale profiling of piRNAs from silkworm ovary and embryos of different developmental stages, we demonstrate that maternally inherited antisense-biased piRNAs trigger acute amplification of secondary sense piRNA production in zygotes, at a time coinciding with zygotic transcription of sense transposon mRNAs. These results provide on-site evidence for the ping-pong cycle.

The comprehensive epigenome map of piRNA clusters

PIWI-interacting RNA (piRNA) clusters act as anti-transposon/retrovirus centers. Integration of selfish jumping elements into piRNA clusters generates de novo piRNAs, which in turn exert trans-silencing activity against these elements in animal gonads. To date, neither genome-wide chromatin modification states of piRNA clusters nor a mode for piRNA precursor transcription have been well understood. Here, to understand the chromatin landscape of piRNA clusters and how piRNA precursors are generated, we analyzed the transcriptome, transcription start sites (TSSs) and the chromatin landscape of the BmN4 cell line, which harbors the germ-line piRNA pathway. Notably, our epigenomic map demonstrated the highly euchromatic nature of unique piRNA clusters. RNA polymerase II was enriched for TSSs that transcribe piRNA precursors. piRNA precursors possessed 5′-cap structures as well as 3′-poly A-tails. Collectively, we envision that the euchromatic, opened nature of unique piRNA clusters or piRNA cluster-associated TSSs allows piRNA clusters to capture new insertions efficiently.

The transcriptional cofactor TRIM33 prevents apoptosis in B lymphoblastic leukemia by deactivating a single enhancer

Most mammalian transcription factors (TFs) and cofactors occupy thousands of genomic sites and modulate the expression of large gene networks to implement their biological functions. In this study, we describe an exception to this paradigm. TRIM33 is identified here as a lineage dependency in B cell neoplasms and is shown to perform this essential function by associating with a single cis element. ChIP-seq analysis of TRIM33 in murine B cell leukemia revealed a preferential association with two lineage-specific enhancers that harbor an exceptional density of motifs recognized by the PU.1 TF. TRIM33 is recruited to these elements by PU.1, yet acts to antagonize PU.1 function. One of the PU.1/TRIM33 co-occupied enhancers is upstream of the pro-apoptotic gene Bim, and deleting this enhancer renders TRIM33 dispensable for leukemia cell survival. These findings reveal an essential role for TRIM33 in preventing apoptosis in B lymphoblastic leukemia by interfering with enhancer-mediated Bim activation. DOI: http://dx.doi.org/10.7554/eLife.06377.001