The dual role of LSD1 and HDAC3 in STAT5-dependent transcription is determined by protein interactions, binding affinities, motifs and genomic positions

STAT5 interacts with other factors to control transcription, and the mechanism of regulation is of interest as constitutive active STAT5 has been reported in malignancies. Here, LSD1 and HDAC3 were identified as novel STAT5a interacting partners in pro-B cells. Characterization of STAT5a, LSD1 and HDAC3 target genes by ChIP-seq and RNA-seq revealed gene subsets regulated by independent or combined action of the factors and LSD1/HDAC3 to play dual role in their activation or repression. Genes bound by STAT5a alone or in combination with weakly associated LSD1 or HDAC3 were enriched for the canonical STAT5a GAS motif, and such binding induced activation or repression. Strong STAT5 binding was see

Specific nuclear envelope transmembrane proteins can promote the location of chromosomes to and from the nuclear periphery

BACKGROUND: Different cell types have distinctive patterns of chromosome positioning in the nucleus. Although ectopic affinity-tethering of specific loci can be used to relocate chromosomes to the nuclear periphery, endogenous nuclear envelope proteins that control such a mechanism in mammalian cells have yet to be widely identified. RESULTS: To search for such proteins twenty three nuclear envelope transmembrane proteins were screened for their ability to promote peripheral localization of human chromosomes in HT1080 fibroblasts. Five of these proteins had strong effects on chromosome 5, but individual proteins affected different subsets of chromosomes. The repositioning effects were reversible and the proteins with effects all exhibited highly tissue-restricted patterns of expression. Depletion of two nuclear envelope transmembrane proteins that were preferentially expressed in liver each reduced the normal peripheral positioning of chromosome 5 in liver cells. CONCLUSIONS: The discovery of nuclear envelope transmembrane proteins that can modulate chromosome position and have restricted patterns of expression may enable dissection of the functional relevance of tissue-specific patterns of radial chromosome positioning.Publisher PDFPeer reviewe

NET23/STING promotes chromatin compaction from the nuclear envelope

Changes in the peripheral distribution and amount of condensed chromatin are observed in a number of diseases linked to mutations in the lamin A protein of the nuclear envelope. We postulated that lamin A interactions with nuclear envelope transmembrane proteins (NETs) that affect chromatin structure might be altered in these diseases and so screened thirty-one NETs for those that promote chromatin compaction as determined by an increase in the number of chromatin clusters of high pixel intensity. One of these, NET23 (also called STING, MITA, MPYS, ERIS, Tmem173), strongly promoted chromatin compaction. A correlation between chromatin compaction and endogenous levels of NET23/STING was observed for a number of human cell lines, suggesting that NET23/STING may contribute generally to chromatin condensation. NET23/STING has separately been found to be involved in innate immune response signaling. Upon infection cells make a choice to either apoptose or to alter chromatin architecture to support focused expression of interferon genes and other response factors. We postulate that the chromatin compaction induced by NET23/STING may contribute to this choice because the cells expressing NET23/STING eventually apoptose, but the chromatin compaction effect is separate from this as the condensation was still observed when cells were treated with Z-VAD to block apoptosis. NET23/STING-induced compacted chromatin revealed changes in epigenetic marks including changes in histone methylation and acetylation. This indicates a previously uncharacterized nuclear role for NET23/STING potentially in both innate immune signaling and general chromatin architecture

Methodologies and genome distribution of the sequences.

<p><b>(A) Scheme of the Methodologies followed:</b> Streptavidin chromatin precipitations and ChIPs followed by streptavidin precipitations were performed in Ba/F3 cells expressing BirA or BirA/bioSTAT5a (left). Single and double ChIPs with anti-STAT5a antibody were performed in Ba/F3 cells (right). All cells were deprived of IL-3 for 6 h and subsequently stimulated with IL-3 for 30 min. <b>(B) Genome distribution of the sequences:</b> Genome distribution of the sequences from the ChIP followed by streptavidin precipitation library is shown on the left and from the double ChIP library on the right. The pie charts show the genome distribution of the sequences relative to the nearest neighbor gene, located within <3 kb and 3–5 kb relative to TSS (transcription start site), introns, exons and intergenic regions.</p

Expression of <i>DPF3</i> gene in patients with hematologic malignancies.

<p>Means of <i>DPF3</i> expression levels (relative mRNA levels), standard errors (SE) and range for each patient cohort and healthy controls are shown. ?Statistical significance refers to comparison with the expression levels in the PB (P₁) or BM (P₂) of healthy controls (Mann-Whitney U test). Statistically significant P values are depicted in bold. Considering that in AML, ALL and CML, a rather similar infiltration of PB and BM by neoplastic cells is observed, a comparison with both groups of healthy controls (PB and BM) was performed.</p

The novel STAT5 target gene <i>Dpf3:</i> Expression levels (in wild type and knock-down cells) and STAT5a, STAT5b binding.

<p><b>(A) Expression levels of <i>Dpf3</i>:</b> Expression levels (mRNA) were measured by real time PCR in Ba/F3 cells deprived of IL-3 for 6 h (starved) and stimulated with IL-3 for 30 min and 3 h. The deprived of IL-3 cells were set as 1. Bars demonstrate mean and standard deviation (SD) values obtained at least in three independent experiments. The statistical significance of the difference in expression between 30 min stimulated and deprived of IL-3 cells is indicated with asterisks (**P = 0.005). <b>(B)</b><b>STAT5a and STAT5b binding to </b><b><i>Dpf3</i></b><b>:</b> Cross-linked chromatin from Ba/F3 cells deprived of IL-3 for 6 h and stimulated with IL-3 for 30 min was used in ChIPs with anti-STAT5a or anti-STAT5b antibody. Three sets of primers were used for each gene. One set specific for the amplification of the isolated genomic region/cloned sequence (seq) containing at least one TTCN_3/4GAA motif, one set specific for the promoter of each gene (pr) containing at least one TTCN₃GAA motif and one set of negative control primers (neg) for amplification of a region lacking TTCN_3/4GAA motifs. IgG was utilized in parallel with anti-STAT5a or anti-STAT5b antibodies, as control. Bars demonstrate mean and SD values of specific enrichments (fold differences) versus input obtained at least in three independent experiments. The statistical significance of enrichment versus the negative control region is indicated with asterisks (ns: not significant, *P = 0.01, **P = 0.002, ***P<0.001). <b>(C)</b><b>Expression levels of </b><b><i>Dpf3</i></b><b> in cells with knock-down of STAT5a, STAT5b or both:</b> Expression levels (mRNA) of <i>Dpf3</i> were measured in Ba/F3 cells with knock-down of STAT5a, STAT5b or both. The cells were deprived of IL-3 for 6 h (starved) and stimulated with IL-3 for 30 min. Expression levels were measured by real time PCR and compared with Ba/F3 cells in the deprived of IL-3 state, transduced with scrambled shRNA and set as 1. Bars demonstrate mean and SD values obtained at least in three independent experiments. The statistical significance of the difference in expression between knock-downs and the respective scrambled control is indicated with asterisks (ns: not significant, *P = 0.02, **P = 0.005, ***P<0.001).</p

STAT5 binding to human <i>DPF3</i> promoter.

<p><b>(A) STAT5a binding to human <i>DPF3</i> promoter in granulocytes:</b> Cross-linked chromatin from granulocytes of a CLL patient and a healthy individual was used in ChIPs with anti-STAT5a antibody. <b>(B) STAT5a binding to human </b><b><i>DPF3</i></b><b> promoter in CLL cell lines:</b> Cross-linked chromatin from EHEB and JVM-2 cells was used in ChIPs with anti-STAT5a antibody. In A and B, IgG was utilized in parallel with anti-STAT5a antibody, as control. Two sets of primers were used: One set specific for the amplification of the <i>DPF3</i> promoter containing a STAT5 motif and one set of negative control primers (neg) for amplification of a region lacking STAT5 motifs. Bars represent mean and SD values of specific enrichments (fold differences) versus input obtained at least in three independent experiments. The statistical significance of enrichment for promoter sequences versus the negative control region (<b>*</b>0.01≤P<0.03) and for patient versus healthy individual (<b>**</b>P = 0.007) is indicated; a-STAT5a: anti-STAT5a.</p

Detection of activated STAT5 (p-STAT5), DPF3 staining and mRNA levels in CLL.

<p><b>(A) Flow cytometry analysis:</b>Indicative plots of peripheral blood cells analysis are shown (healthy individual with low <i>DPF3</i> expression levels (3.6) in upper panels and CLL patient with high <i>DPF3</i> expression levels (19.69) in lower panels). Dot plots on the left indicate percentages of gated cells used on the analysis (A: lymphocytes in pink, B: monocytes in green and C: granulocytes in red). Histograms on the right indicate p-STAT5 levels in lymphocytes (pink), monocytes (green) and granulocytes (red). Isotype control (IgG1) is depicted in grey. <b>(B) Levels of p-STAT5 in the peripheral blood cell subpopulations analyzed by flow cytometry:</b> The boxes represent the interquartile range containing 50% of values in lymphocytes, monocytes and granulocytes of healthy individuals (n = 4) and CLL patients (n = 10). The whiskers are lines that extend from the box to the highest and lowest values, excluding outliers. A line across the box indicates the median value for each group. The statistical significance of difference is noted on the charts. <b>(C) </b><b><i>DPF3</i></b><b> expression levels (mRNA) in granulocytes and monocytes: </b><i>DPF3</i> expression levels were measured in granulocytes and monocytes of CLL patients (n = 5) and healthy individuals (n = 4) by real time PCR. Bars demonstrate mean and standard error of the mean (SEM) values of expression. The statistical significance of the differences in expression is indicated with an asterisk (*0.01≤P<0.05). <b>(D) Immunofluorescence detection of nuclear p-STAT5:</b> Granulocytes with low to high intensity nuclear p-STAT5 staining were scored as positive for the nucleus. Granulocytes with absent nuclear staining and low to high intensity cytoplasmic staining were considered positive for the cytoplasm (***P<0.001). <b>(E) Immunofluorescence detection of nuclear DPF3:</b> Scoring of nuclear DPF3 staining (detected as puncta in the nucleus) was performed on granulocytes positive for p-STAT5 staining in the cytoplasm and/or the nucleus. Granulocytes with low to high intensity puncta in the nucleus were considered positive (***P<0.001). <b>(F) Immunofluorescence detection of both nuclear p-STAT5 and DPF3:</b> For co-localization of DPF3 (red) and p-STAT5 (green) nuclear staining, granulocytes with green and red, and/or yellow staining puncta (representing p-STAT5 and DPF3 co-localization) were scored (*P = 0.05). In D, E and F: Scoring was performed in granulocytes showing p-STAT5 staining in the nucleus, the cytoplasm or both; bars demonstrate mean and SD values obtained at least in three different slides or regions of the same slide; the statistical significance of the difference between samples with low (healthy individuals) and high (CLL patients) <i>DPF3</i> expression is shown; <i>DPF3</i> expression levels (mRNA) are shown below the samples; ND: not determined.</p