the others (reviewed by Kelley and Kuroda, 1995). This observation suggests that these gene products must interact Dosage compensation is a regulatory process that and form a complex. Preliminary direct evidence for such insures that males and females have equal amounts of an interaction is provided by the observation that MSL-1 X-chromosome gene products. In Drosophila, this is and MSL-2 can be co-precipitated with antisera against achieved by a 2-fold enhancement of X-linked gene either protein (Kelley et al., 1995). Binding of MLE and transcription in males, relative to females. The the MSL proteins to the X chromosome in males is enhancement of transcription is mediated by the correlated with the appearance of histone 4 acetylated at activity of a group of regulatory genes characterized Lys16 (H4Ac16) on the same chromosome (Turner et al., by the male-specific lethality of their loss-of-function 1992) and at the same sites (Bone et al., 1994). alleles. The products of these genes form a complexWe have used the phenotype of male-specific lethality that is preferentially associated with numerous sites on to screen the X chromosome of Drosophila melanogaster the X chromosome in somatic cells of males but not of for ethyl methane sulfonate (EMS)-induced mutations, females. Binding of the dosage compensation complex identifying additional genes that may be involved in the is correlated with a significant increase in the presence regulatory process of dosage compensation. We isolated of a specific histone isoform, histone 4 acetylated at one such mutation (mof) and observed that dying mutant Lys16, on this chromosome. Experimental results and males lack the X-associated isoform of H4Ac16. MOF sequence analysis suggest that an additional gene, exhibits the signature motif for the acetyl coenzyme males-absent on the first (mof), encodes a putative acetyl A binding site found in numerous and diverse acetyl transferase that plays a direct role in the specific histone transferases, and the mof mutation is a single amino acid acetylation associated with dosage compensation. The substitution in the most conserved residue of this motif. predicted amino acid sequence of MOF exhibits a This provides evidence that MOF is the histone acetyl significant level of similarity to several other proteins, transferase (HAT) responsible for the particular histone including the human HIV-1 Tat interactive protein acetylation involved in the male-specific hypertranscripTip60, the human monocytic leukemia zinc finger tion of X-linked genes. protein MOZ and the yeast silencing proteins SAS3 and SAS2.

The cellular heterogeneity of neoplasms has been at the center of considerable interest since the "cancer stem cell hypothesis", originally formulated for hematologic malignancies, was extended to solid tumors.

Posttranslational acetylation of core histone amino termini has long been associated with transcriptionally active chromatin. Recent reports have demonstrated histone acetyltransferase activity in a small group of conserved transcriptional regulators directly linked to gene activation. In addition, the presence of a putative acetyltransferase domain has been discovered in a group of proteins known as the MYST family (for its founding members MOZ, YBF2͞ SAS3, SAS2, and Tip60). Members of this family are implicated in acute myeloid leukemia (MOZ), transcriptional silencing in yeast (SAS2 and YBF2͞SAS3), HIV Tat interaction in humans (Tip60), and dosage compensation in Drosophila (MOF). In this report, we express a yeast ORF with homology to MYST family members and show it possesses histone acetyltransferase activity. Unlike the other MYST family members in Saccharomyces cerevisiae this gene is essential for growth.

In Drosophila, dosage compensation-the equalization of most X-linked gene products in males and females-is achieved by a twofold enhancement of the level of transcription of the X chromosome in males relative to each X chromosome in females. A complex consisting of at least five gene products preferentially binds the X chromosome at numerous sites in males and results in a significant increase in the presence of a specific histone isoform, histone 4 acetylated at lysine 16. Recently, RNA transcripts (roX1 and roX2) encoded by two different genes have also been found associated with the X chromosome in males. We have partially purified a complex containing MSL1, -2, and -3, MOF, MLE, and roX2 RNA and demonstrated that it exclusively acetylates H4 at lysine 16 on nucleosomal substrates. These results demonstrate that the MSL complex is responsible for the specific chromatin modification characteristic of the X chromosome in Drosophila males.Dosage compensation is a regulatory mechanism to ensure that the level of expression of genes on the single X chromosome of Drosophila males equals the level attained from the two X chromosomes in females. This equalization, achieved by a twofold increase in the rate of X-linked gene transcription in males relative to females, has been observed for a wide variety of genes with promoters of different strengths, in many cell types, and at different developmental stages. For this reason, the study of dosage compensation may provide valuable insights into the mechanisms that regulate levels of transcription.Five genes involved in dosage compensation have been identified based on the male-specific lethality of their loss-of-function alleles (26). The products of these genes, collectively referred to as MSL proteins, colocalize to the male X chromosome, a chromosome that is also highly enriched with histone H4 acetylated at lysine 16 (6, 41). Since in all eukaryotes acetylation of the histones has been correlated directly with the establishment and regulation of transcription (reviewed in reference 28), it is likely that the MSL complex mediates its effect, at least in part, through histone acetylation. Indeed, the most recent MSL to be discovered is MOF (for "males absent on the first"), a protein with homology to acetyltransferases of the MYST family (8,18,33).Another protein component of the complex is MLE (for "maleless"), an ATP-dependent RNA or DNA helicase (25). Unlike the other members of the MSL complex, MLE can be dissociated from the X chromosome by treatment with RNase, suggesting that the complex may interact with either nascent or some other form of RNA (34). This speculation has been reinforced, if not validated, by the recent discovery of two genes, roX1 and roX2 (for "RNA on the X 1 and 2") that encode RNAs with no apparent open reading frames (1, 27). These RNAs are found only in males, and their presence depends on the MSL complex, with which they are seen to colocalize on the X chromosome (15,20).In this paper, we report the initial functional characterization of ...

In Drosophila the equalization of X‐linked gene products between males and females, i.e. dosage compensation, is the result of a 2‐fold hypertranscription of most of these genes in males. At least four regulatory genes are required for this process. Three of these genes, maleless (mle), male‐specific lethal 1 (msl‐1) and male‐specific lethal 3 (msl‐3), have been cloned and their products have been shown to interact and to bind to numerous sites on the X chromosome of males, but not of females. Although binding to the X chromosome is negatively correlated with the function of the master regulatory gene Sex lethal (Sxl), the mechanisms that restrict this binding to males and to the X chromosome are not yet understood. We have cloned the last of the known autosomal genes involved in dosage compensation, male‐specific lethal 2 (msl‐2), and characterized its product. The encoded protein (MSL‐2) consists of 769 amino acid residues and has a RING finger (C3HC4 zinc finger) and a metallothionein‐like domain with eight conserved and two non‐conserved cysteines. In addition, it contains a positively and a negatively charged amino acid residue cluster and a coiled coil domain that may be involved in protein‐protein interactions. Males produce a msl‐2 transcript that is shorter than in females, due to differential splicing of an intron of 132 bases in the untranslated leader. Using an antiserum against MSL‐2 we have shown that the protein is expressed at a detectable level only in males, where it is physically associated with the X chromosome. Our observations suggest that MSL‐2 may be the target of the master regulatory gene Sxl and provide the basic elements of a working hypothesis on the function of MSL‐2 in mediating the 2‐fold increase in transcription that is characteristic of dosage compensation.

Abstractγ‐Secretase inhibitors (GSIs) are being actively repurposed as cancer therapeutics based on the premise that inhibition of NOTCH1 signaling in select cancers is therapeutic. Using novel assays to probe effects of GSIs against a broader panel of substrates, we demonstrate that clinical GSIs are pharmacologically distinct. GSIs show differential profiles of inhibition of the various NOTCH substrates, with some enhancing cleavage of other NOTCH substrates at concentrations where NOTCH1 cleavage is inhibited. Several GSIs are also potent inhibitors of select signal peptide peptidase (SPP/SPPL) family members. Extending these findings to mammosphere inhibition assays in triple‐negative breast cancer lines, we establish that these GSIs have different functional effects. We also demonstrate that the processive γ‐secretase cleavage pattern established for amyloid precursor protein (APP) occurs in multiple substrates and that potentiation of γ‐secretase cleavage is attributable to a direct action of low concentrations of GSIs on γ‐secretase. Such data definitively demonstrate that the clinical GSIs are not biological equivalents, and provide an important framework to evaluate results from ongoing and completed human trials with these compounds.

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Approximately 80% of breast cancers express the estrogen receptor-α (ERα) and are treated with anti-estrogens. Resistance to these agents is a major cause of mortality. We have shown that estrogen inhibits Notch, whereas anti-estrogens or estrogen withdrawal activate Notch signaling. Combined inhibition of Notch and estrogen signaling has synergistic effects in ERα-positive breast cancer models. However, the mechanisms whereby Notch-1 promotes the growth of ERα-positive breast cancer cells are unknown. Here, we demonstrate that Notch-1 increases the transcription of ERα-responsive genes in the presence or absence of estrogen via a novel chromatin crosstalk mechanism. Our data support a model in which Notch-1 can activate the transcription of ERα-target genes via IKKα-dependent cooperative chromatin recruitment of Notch–CSL–MAML1 transcriptional complexes (NTC) and ERα, which promotes the recruitment of p300. CSL binding elements frequently occur in close proximity to estrogen-responsive elements (EREs) in the human and mouse genomes. Our observations suggest that a hitherto unknown Notch-1/ERα chromatin crosstalk mediates Notch signaling effects in ERα-positive breast cancer cells and contributes to regulate the transcriptional functions of ERα itself.