In animals, PIWI-interacting RNAs (piRNAs) guide PIWI proteins to silence transposons and regulate gene expression. The mechanisms for making piRNAs have been proposed to differ among cell types, tissues, and animals. Our data instead suggest a single model that explains piRNA production in most animals. piRNAs initiate piRNA production by guiding PIWI proteins to slice precursor transcripts. Next, PIWI proteins direct the stepwise fragmentation of the sliced precursor transcripts, yielding tail-to-head strings of phased precursor piRNAs (pre-piRNAs). Our analyses detect evidence for this piRNA biogenesis strategy across an evolutionarily broad range of animals, including humans. Thus, PIWI proteins initiate and sustain piRNA biogenesis by the same mechanism in species whose last common ancestor predates the branching of most animal lineages. The unified model places PIWI-clade Argonautes at the center of piRNA biology and suggests that the ancestral animal-the Urmetazoan-used PIWI proteins both to generate piRNA guides and to execute piRNA function.

Mycobacterium tuberculosis (Mtb) survives within macrophages by evading delivery to the lysosome and promoting the accumulation of lipid bodies, which serve as a bacterial source of nutrients. Here we show that by inducing miR-33 and its passenger strand miR-33*, Mtb inhibits integrated pathways involved in autophagy, lysosomal function and fatty acid oxidation to support bacterial replication. Silencing of miR-33 and miR-33* by genetic or pharmacological means promotes autophagy flux through derepression of key autophagy effectors such as ATG5, ATG12, LC3B and LAMP1 and AMPK-dependent activation of the transcription factors FOXO3 and TFEB, enhancing lipid catabolism and Mtb xenophagy. These data define a mammalian miRNA circuit utilized by Mtb to coordinately inhibit autophagy and reprogram host lipid metabolism to enable intracellular survival and persistence in the host.

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Pachytene piRNAs, which comprise >80% of small RNAs in the adult mouse testis, have been proposed to bind and regulate target RNAs like miRNAs, cleave targets like siRNAs, or lack biological function altogether. Although piRNA pathway protein mutants are male sterile, no biological function has been identified for any mammalian piRNA-producing locus. Here, we report that males lacking piRNAs from a conserved mouse pachytene piRNA locus on chromosome 6 ( pi6 ) produce sperm with defects in capacitation and egg fertilization. Moreover, heterozygous embryos sired by pi6 −/− fathers show reduced viability in utero. Molecular analyses suggest that pi6 piRNAs repress gene expression by cleaving mRNAs encoding proteins required for sperm function, pi6 also participates in a network of piRNA-piRNA precursor interactions that initiate piRNA production from a second piRNA locus on chromosome 10 as well as pi6 itself. Our data establish a direct role for pachytene piRNAs in spermiogenesis and embryo viability.

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Establishment and segregation of distinct chromatin domains are essential for proper genome function. The insulator protein CCCTC-binding factor (CTCF) is involved in creating boundaries that segregate chromatin and functional domains and in organizing higher-order chromatin structures by promoting chromosomal loops across the vertebrate genome. Here, we investigate the insulation properties of CTCF at the human and mouse homeobox gene A ( HOXA ) loci. Although cohesin loading at the CTCF binding site is required for looping, we found that cohesin is dispensable for chromatin barrier activity at that site. Using mouse embryonic stem cells in both a pluripotent and differentiated neuronal progenitor state, we determined that embryonic stem cell pluripotency factor OCT4 antagonizes cohesin loading at the CTCF binding site. Loss of OCT4 in the committed and differentiated neuronal progenitor cells results in loading of cohesin and chromosome looping, which contributes to heterochromatin partitioning and selective gene activation across the HOXA locus. Our analysis reveals that chromatin barrier activity of CTCF is evolutionarily conserved and is responsible for the coordinated establishment of chromatin structure, higher-order architecture, and developmental expression of the HOXA locus.

Treatment with histone deacetylase inhibitors (HDACI) results in potent cytotoxicity of a variety of cancer cell types, and these drugs are used clinically to treat hematological tumors. They are known to repress the transcription of ERBB2 and many other oncogenes, but little is known about this mechanism. Using global run-on sequencing (GRO-seq) to measure nascent transcription, we find that HDACI cause transcriptional repression by blocking RNA polymerase II elongation. Our data show that HDACI preferentially repress the transcription of highly expressed genes as well as high copy number genes in HER2+ breast cancer genomes. In contrast, genes that are activated by HDACI are moderately expressed. We analyzed gene copy number in combination with microarray and GRO-seq analysis of expression level, in normal and breast cancer cells to show that high copy number genes are more likely to be repressed by HDACI than non-amplified genes. The inhibition of transcription of amplified oncogenes, which promote survival and proliferation of cancer cells, might explain the cancer-specific lethality of HDACI, and may represent a general therapeutic strategy for cancer.

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