Bromodomain protein BRD4 is a transcriptional repressor of autophagy and lysosomal function

Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation

Emerging roles of transcriptional programs in autophagy regulation

Autophagy is an essential cellular process that degrades cytoplasmic organelles and components. Precise control of autophagic activity is achieved by context-dependent signaling pathways. Recent studies have highlighted the involvement of transcriptional programs during autophagic responses to various signals. Here, we summarize the current understanding of the transcriptional regulation of autophagy

Msh2 acts in medium-spiny striatal neurons as an enhancer of CAG instability and mutant huntingtin phenotypes in Huntington's disease knock-in mice.

The CAG trinucleotide repeat mutation in the Huntington's disease gene (HTT) exhibits age-dependent tissue-specific expansion that correlates with disease onset in patients, implicating somatic expansion as a disease modifier and potential therapeutic target. Somatic HTT CAG expansion is critically dependent on proteins in the mismatch repair (MMR) pathway. To gain further insight into mechanisms of somatic expansion and the relationship of somatic expansion to the disease process in selectively vulnerable MSNs we have crossed HTT CAG knock-in mice (HdhQ111) with mice carrying a conditional (floxed) Msh2 allele and D9-Cre transgenic mice, in which Cre recombinase is expressed specifically in MSNs within the striatum. Deletion of Msh2 in MSNs eliminated Msh2 protein in those neurons. We demonstrate that MSN-specific deletion of Msh2 was sufficient to eliminate the vast majority of striatal HTT CAG expansions in HdhQ111 mice. Furthermore, MSN-specific deletion of Msh2 modified two mutant huntingtin phenotypes: the early nuclear localization of diffusely immunostaining mutant huntingtin was slowed; and the later development of intranuclear huntingtin inclusions was dramatically inhibited. Therefore, Msh2 acts within MSNs as a genetic enhancer both of somatic HTT CAG expansions and of HTT CAG-dependent phenotypes in mice. These data suggest that the selective vulnerability of MSNs may be at least in part contributed by the propensity for somatic expansion in these neurons, and imply that intervening in the expansion process is likely to have therapeutic benefit

Deletion of <i>Msh2</i> in medium-spiny striatal neurons eliminates the majority of striatal <i>HTT</i> CAG expansions.

<p>GeneMapper traces of PCR-amplified <i>HTT</i> CAG repeats from striatum, cortex, liver and tail DNA of representative five-month <i>HdhQ111</i>/+ mice (<b>A</b>) or from striatum and tail of representative ten month <i>HdhQ111</i>/+ mice (<b>C</b>) with <i>Msh2</i>+/+, <i>Msh2</i>+/−, <i>Msh2Δ</i>/<i>Δ</i>, <i>Msh2Δ</i>/− and <i>Msh2</i>−/− genotypes. Constitutive CAG repeat lengths, as determined in tail DNA, are indicated. Instability indices were quantified from GeneMapper traces of PCR-amplified <i>HTT</i> CAG repeats from five-month striatum, cortex and liver (<b>B</b>) and ten-month striatum (<b>D</b>) of <i>HdhQ111</i>/+ mice with <i>Msh2</i>+/+, <i>Msh2</i>+/−, <i>Msh2Δ</i>/<i>Δ</i>, <i>Msh2Δ</i>/− and <i>Msh2</i>−/−genotypes. Five-month mice: <i>Msh2</i>+/+ (n = 6, CAG 113, 118, 119, 121, 123, 125), <i>Msh2</i>+/− (n = 4, CAG 114, 114, 120, 123), <i>Msh2Δ</i>/<i>Δ</i>(n = 5, CAG 113, 121, 121, 126, 129), <i>Msh2Δ</i>/−(n = 7, CAG 113, 121, 121, 122, 125, 125, 133) and <i>Msh2</i>−/− (n = 3, CAG 112, 120, 123). Ten-month mice: <i>Msh2</i>+/+ (n = 6, CAG 118, 121, 121, 123, 126, 134), <i>Msh2</i>+/− (n = 4, CAG 116, 118, 123, 131), <i>Msh2Δ</i>/<i>Δ</i> (n = 1, CAG 133), <i>Msh2Δ</i>/− (n = 7, CAG 115, 115, 117, 120, 121, 122, 123) and <i>Msh2</i>−/− (n = 1, CAG 132). Bars represent mean ± S.D. *** p<0.0001, * p<0.05 (Student’s t-test).</p

Conditional deletion of the <i>floxed Msh2</i> allele in the striatum. A.

<p>Genotyping for the conditional <i>Msh2</i> allele in genomic DNA extracted from striatum of <i>Msh2</i>+/+, <i>Msh2flox</i>/+, <i>Msh2flox</i>/+ <i>D9-Cre</i> and <i>Msh2flox</i>/flox <i>D9-Cre</i> mice. The deleted (Δ) <i>Msh2</i> allele is present only in mice harboring both the <i>Msh2flox</i> allele and the <i>D9-Cre</i> transgene. <b>B.</b> Genotyping for the conditional <i>Msh2</i> allele in genomic DNA extracted from five different tissues from a <i>Msh2flox</i>/+ <i>D9-Cre</i> mouse shows that the deletion is specific for the striatum. Mice were six weeks of age. <i>flox</i>: <i>Msh2</i> allele flanked by <i>loxP</i> sites; Δ:deleted <i>Msh2</i> allele; wt: wild-type <i>Msh2</i> allele.</p

Deletion of Msh2 in medium-spiny neurons delays nuclear huntingtin phenotypes. A, B.

<p>Nuclear mutant huntingtin immunostaining is decreased in the striata of five-month old <i>HdhQ111</i>/+ mice with deletion of <i>Msh2</i> in MSNs. <b>A.</b> Fluorescent micrographs of striata double-stained with anti-huntingtin mAb5374 and anti-histone H3 antibodies for three CAG repeat length-matched mice (<i>Msh2</i>+/+ CAG 113, <i>Msh2Δ</i>/<i>Δ</i> CAG 112, <i>Msh2−/−</i> CAG 113). <b>B.</b> Box plot showing upper and lower quartiles, median and range for the normalized mAb5374 immunostaining intensity (total mAb5374 staining intensity normalized to the number of H3-positive nuclei). Outlier (circle) is defined by a standard interquartile method and is included in the analysis. Multiple regression analysis was used to determine the effect of <i>Msh2</i> genotype on mAb5374 staining using normalized mAb5374 intensity (continuous variable) as a dependent variable and <i>Msh2</i> genotype (discrete variable), constitutive CAG length (continuous variable) and position (medial versus lateral, discrete variable) as independent variables. Both constitutive CAG length (P<0.05) and medial versus lateral position (P<0.001) were significantly associated with normalized mAb5374 intensity. Asterisks above the bars indicate a significant difference from <i>Msh2</i>+/+ at a p-value cut-off of p<0.05(*), p<0.01 (**), p<0.001 (***) in the regression analysis. <i>Msh2Δ</i>/− was not significantly different from <i>Msh2</i>+/− (p = 0.18). The five-month mice used in the quantitative analysis are as follows: <i>Msh2</i>+/+ (n = 6, CAG 113, 118, 119, 121, 123, 125), <i>Msh2</i>+/− (n = 4, CAG 114, 114, 120, 123), <i>Msh2Δ</i>/<i>Δ</i> (n = 5, CAG 113, 121, 121, 126, 129), <i>Msh2Δ</i>/− (n = 7, CAG 113, 121, 121, 122, 125, 125, 133) and <i>Msh2</i>−/− (n = 3, CAG 112, 120, 123). Note that the relatively “weak” effect of the <i>Msh2</i>−/− genotype likely reflects the small number of mice of this genotype and hence the least accurate estimate of the relationship of mAb5374 intensity to CAG length in the regression analysis. <b>C, D.</b> Intranuclear inclusions are decreased in the striata of ten-month old <i>HdhQ111</i>/+ mice with deletion of <i>Msh2</i> in MSNs. <b>C.</b> Fluorescent micrographs of striata stained with mAb5374 from mice with <i>Msh2</i>+/+ (CAG 121), <i>Msh2</i>+/− (CAG 123), <i>Msh2Δ</i>/<i>Δ</i> (CAG 133), <i>Msh2Δ</i>/− (CAG 123) and <i>Msh2</i>−/− (CAG 132) genotypes. <b>D.</b> Quantification of the percentage of cells containing an inclusion (more than one inclusion per cell was rarely observed). The total number of cells was determined by co-staining with histone H3 (not shown). The ten-month mice used in the quantitative analysis are as follows: <i>Msh2</i>+/+ (n = 6, CAG 118, 121, 121, 123, 126, 134), <i>Msh2</i>+/− (n = 4, CAG 116, 118, 123, 131), <i>Msh2Δ</i>/<i>Δ</i> (n = 1, CAG 133), <i>Msh2Δ</i>/− (n = 7, CAG 115, 115, 117, 120, 121, 122, 123) and <i>Msh2</i>−/− (n = 1, CAG 132). Bars represent mean ±S.D.</p