Plasma HDL levels have a protective role in atherosclerosis, yet clinical therapies to raise HDL levels have remained elusive. Recent advances in the understanding of lipid metabolism have revealed that miR-33, an intronic microRNA located within the SREBF2 gene, suppresses expression of the cholesterol transporter ABC transporter A1 (ABCA1) and lowers HDL levels. Conversely, mechanisms that inhibit miR-33 increase ABCA1 and circulating HDL levels, suggesting that antagonism of miR-33 may be atheroprotective. As the regression of atherosclerosis is clinically desirable, we assessed the impact of miR-33 inhibition in mice deficient for the LDL receptor (Ldlr -/-mice), with established atherosclerotic plaques. Mice treated with anti-miR33 for 4 weeks showed an increase in circulating HDL levels and enhanced reverse cholesterol transport to the plasma, liver, and feces. Consistent with this, anti-miR33-treated mice showed reductions in plaque size and lipid content, increased markers of plaque stability, and decreased inflammatory gene expression. Notably, in addition to raising ABCA1 levels in the liver, anti-miR33 oligonucleotides directly targeted the plaque macrophages, in which they enhanced ABCA1 expression and cholesterol removal. These studies establish that raising HDL levels by anti-miR33 oligonucleotide treatment promotes reverse cholesterol transport and atherosclerosis regression and suggest that it may be a promising strategy to treat atherosclerotic vascular disease.

Scarring of the kidney is a major public health concern, directly promoting loss of kidney function. In order to understand the role of microRNA (miRNA) in the progression of kidney scarring in response to injury, we investigated changes in miRNA expression in two kidney fibrosis models, and identified 24 commonly upregulated miRNAs. Among them, miR-21 was highly elevated in both animal models and human transplant kidney nephropathy. Deletion of miR-21 in mice resulted in no overt abnormality. However, miR-21-/- mice suffered far less interstitial fibrosis in response to kidney injury, which was pheno-copied in wild-type mice treated with anti-miR-21 oligonucleotides. Surprisingly, global de-repression of miR-21 target messenger RNAs was only readily detectable in miR-21-/- kidneys after injury. Analysis of gene expression profiles identified groups of genes involved in metabolic pathways that were up-regulated in the absence of miR-21, including the lipid metabolism pathway regulated by Peroxisome proliferator activated receptor-α (Pparα), a direct miR-21 target. Over-expression of Pparα prevented UUO-induced injury and fibrosis. Pparα deficiency abrogated the anti-fibrotic effect of anti-miR21 oligonucleotides. miR-21 also regulates the redox metabolic pathway. The mitochondrial inhibitor of reactive oxygen species generation, Mpv17l, was repressed by miR-21, correlating closely with enhanced oxidative kidney damage. These studies demonstrate that miR-21 contributes to fibrogenesis and epithelial injury in the kidney in two mouse models and is a candidate target for anti-fibrotic therapies.

We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbations coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with dual-guide RNAs in three cell lines, altogether comprising 141,912 tests of interaction. Numerous therapeutically relevant interactions were identified and these patterns replicated with combinatorial drugs at 75% precision. Based on these results we anticipate cellular context will be critical to synthetic-lethal therapies.

The hypoxia-inducible factor (HIF) pathway is essential for cell survival under low oxygen and plays an important role in tumor cell homeostasis. We investigated the function of miR-210, the most prominent microRNA upregulated by hypoxia and a direct transcriptional target of HIFs. miR-210 expression was elevated in multiple cancer types and correlated with metastasis of breast and melanoma tumors. miR-210 overexpression in cancer cell lines bypassed hypoxia-induced cell cycle arrest and partially reversed the hypoxic gene expression signature. We identified MNT, a known MYC antagonist, as a miR-210 target. MNT mRNA contains multiple miR-210 binding sites in the 3' UTR and its knockdown phenocopied miR-210 overexpression. Furthermore, loss of MYC abolished miR-210-mediated override of hypoxia-induced cell cycle arrest. Comparison of miR-210 and MYC overexpression with MNT knockdown signatures also indicated that miR-210 triggered a "MYC-like" transcriptional response. Thus, miR-210 influences the hypoxia response in tumor cells through targeting a key transcriptional repressor of the MYC-MAX network.

Cell cycle arrest in response to DNA damage is an important antitumorigenic mechanism. MicroRNAs (miRNAs) were recently shown to play key regulatory roles in cell cycle progression. For example, miR-34a is induced in response to p53 activation and mediates G 1 arrest by down-regulating multiple cell cycle-related transcripts. Here we show that genotoxic stress promotes the p53-dependent up-regulation of the homologous miRNAs miR-192 and miR-215. Like miR-34a, activation of miR-192/215 induces cell cycle arrest, suggesting that multiple miRNA families operate in the p53 network. Furthermore, we define a downstream gene expression signature for miR-192/215 expression, which includes a number of transcripts that regulate G 1 and G 2 checkpoints. Of these transcripts, 18 transcripts are direct targets of miR-192/ 215, and the observed cell cycle arrest likely results from a cooperative effect among the modulations of these genes by the miRNAs. Our results showing a role for miR-192/215 in cell proliferation combined with recent observations that these miRNAs are underexpressed in primary cancers support the idea that miR-192 and miR-215 function as tumor suppressors. [Cancer Res 2008;68(24):10105-12]

SUMMARY It is now established that Bcl11b specifies T cell fate. Here we show that in developing T-cells the Bcl11b enhancer repositioned from the lamina to the nuclear interior. Our search for factors that relocalized the Bcl11b enhancer identified a non-coding RNA named ThymoD (Thymocyte Differentiation Factor). ThymoD-deficient mice displayed a block at the onset of T cell development and developed lymphoid malignancies. We found that ThymoD transcription promoted demethylation at CTCF bound sites and activated cohesin-dependent looping to reposition the Bcl11b enhancer from the lamina to the nuclear interior and to juxtapose the Bcl11b enhancer and promoter into a single loop domain. These large-scale changes in nuclear architecture were associated with the deposition of activating epigenetic marks across the loop domain, plausibly facilitating phase separation. These data indicate how during developmental progression and tumor suppression non-coding transcription orchestrates chromatin folding and compartmentalization to direct with high precision enhancer-promoter communication.

MYC oncogene family members are broadly implicated in human cancers, yet are considered "undruggable" as they encode transcription factors. MYC also carries out essential functions in proliferative tissues, suggesting that its inhibition could cause severe side effects. We elected to identify synthetic lethal interactions with c-MYC overexpression (MYC-SL) in a collection of ∼3,300 druggable genes, using high-throughput siRNA screening. Of 49 genes selected for follow-up, 48 were confirmed by independent retesting and approximately one-third selectively induced accumulation of DNA damage, consistent with enrichment in DNA-repair genes by functional annotation. In addition, genes involved in histone acetylation and transcriptional elongation, such as TRRAP and BRD4, were identified, indicating that the screen revealed known MYC-associated pathways. For in vivo validation we selected CSNK1e, a kinase whose expression correlated with MYCN amplification in neuroblastoma (an established MYC-driven cancer). Using RNAi and available small-molecule inhibitors, we confirmed that inhibition of CSNK1e halted growth of MYCN-amplified neuroblastoma xenografts. CSNK1e had previously been implicated in the regulation of developmental pathways and circadian rhythms, whereas our data provide a previously unknown link with oncogenic MYC. Furthermore, expression of CSNK1e correlated with c-MYC and its transcriptional signature in other human cancers, indicating potential broad therapeutic implications of targeting CSNK1e function. In summary, through a functional genomics approach, pathways essential in the context of oncogenic MYC but not to normal cells were identified, thus revealing a rich therapeutic space linked to a previously "undruggable" oncogene.

The function of GATA transcription factors in diverse developmental contexts depends in part on physical interaction with cofactors of the Friend of GATA (FOG) family. However, previous studies indicate that FOG-1 may play a GATA-1-independent role in early megakaryopoiesis, suggesting that FOG proteins might act in a GATA factor-independent manner. Here, we have generated mouse knock-in (KI) mutants harboring a critical valine-to-glycine substitution in the amino-terminal zinc fingers of GATA-1 and GATA-2 to ablate FOG interaction. In contrast to male GATA-1 KI (GATA-1 is located on the X-chromosome) or GATA-2 KI/KI mice, compound GATA-1 KI GATA-2 KI/KI mutant mice display complete megakaryopoietic failure, a phenocopy of FOG-1 ؊/؊ mice. We conclude that FOG-1 requires an interaction with either GATA-1 or -2 as part of its essential role in early megakaryopoiesis. On the basis of these and previous reports, we infer that GATA factor dependence is a critical aspect of FOG protein function.