Rui Fang

Loss of 5-Hydroxymethylcytosine Is an Epigenetic Hallmark of Melanoma

DNA methylation at the 5 position of cytosine (5-mC) is a key epigenetic mark that is critical for various biological and pathological processes. 5-mC can be converted to 5-hydroxymethylcytosine (5-hmC) by the ten-eleven translocation (TET) family of DNA hydroxylases. Here, we report that loss of 5-hmC is an epigenetic hallmark of melanoma, with diagnostic and prognostic implications. Genome-wide mapping of 5-hmC reveals loss of the 5-hmC landscape in the melanoma epigenome. We show that downregulation of isocitrate dehydrogenase 2 (IDH2) and TET family enzymes is likely one of the mechanisms underlying 5-hmC loss in melanoma. Rebuilding the 5-hmC landscape in melanoma cells by reintroducing active TET2 or IDH2 suppresses melanoma growth and increases tumor-free survival in animal models. Thus, our study reveals a critical function of 5-hmC in melanoma development and directly links the IDH and TET activity-dependent epigenetic pathway to 5-hmC-mediated suppression of melanoma progression, suggesting a new strategy for epigenetic cancer therapy.

The histone H3K4 demethylase SMCX links REST target genes to X-linked mental retardation

Gene transcription is critically influenced by chromatin structure and the modification status of histone tails. Methylation of lysine residues in histone tails is dynamically regulated by the opposing activities of histone methyltransferases and histone demethylases. Here we show that JARID1C/SMCX, a JmjC-domain-containing protein implicated in X-linked mental retardation and epilepsy, possesses H3K4 tri-demethylase activity and functions as a transcriptional repressor. An SMCX complex isolated from HeLa cells contains additional chromatin modifiers (the histone deacetylases HDAC1 and HDAC2, and the histone H3K9 methyltransferase G9a) and the transcriptional repressor REST, suggesting a direct role for SMCX in chromatin dynamics and REST-mediated repression. Chromatin immunoprecipitation reveals that SMCX and REST co-occupy the neuron-restrictive silencing elements in the promoters of a subset of REST target genes. RNA-interference-mediated depletion of SMCX derepresses several of these targets and simultaneously increases H3K4 trimethylation at the sodium channel type 2A (SCN2A) and synapsin I (SYN1) promoters. We propose that loss of SMCX activity impairs REST-mediated neuronal gene regulation, thereby contributing to SMCX-associated X-linked mental retardation.

High-resolution Xist binding maps reveal two-step spreading during X-chromosome inactivation

The Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation (XCI), the process by which mammals compensate for unequal numbers of sex chromosomes. During XCI, Xist coats the future inactive X chromosome (Xi) and recruits Polycomb repressive complex 2 (PRC2) to the X-inactivation centre (Xic). How Xist spreads silencing on a 150-megabases scale is unclear. Here we generate high-resolution maps of Xist binding on the X chromosome across a developmental time course using CHART-seq. In female cells undergoing XCI de novo, Xist follows a two-step mechanism, initially targeting gene-rich islands before spreading to intervening gene-poor domains. Xist is depleted from genes that escape XCI but may concentrate near escapee boundaries. Xist binding is linearly proportional to PRC2 density and H3 lysine 27 trimethylation (H3K27me3), indicating co-migration of Xist and PRC2. Interestingly, when Xist is acutely stripped off from the Xi in post-XCI cells, Xist recovers quickly within both gene-rich and gene-poor domains on a timescale of hours instead of days, indicating a previously primed Xi chromatin state. We conclude that Xist spreading takes distinct stage-specific forms. During initial establishment, Xist follows a two-step mechanism, but during maintenance, Xist spreads rapidly to both gene-rich and gene-poor regions.

Human LSD2/KDM1b/AOF1 regulates gene transcription by modulating intragenic H3K4me2 methylation.

Dynamic histone H3K4 methylation is an important epigenetic component of transcriptional regulation. However, most of our current understanding of this histone mark is confined to the regulation of transcriptional initiation. We now show that human LSD2/KDM1b/AOF1, the human homolog of LSD1, is an H3K4me1/2 demethylase that specifically regulates histone H3K4 methylation within intragenic regions of its target genes. Genome-wide mapping reveals that LSD2 associates predominantly with the gene bodies of actively transcribed genes, but is markedly absent from promoters. Depletion of endogenous LSD2 results in an increase of H3K4me2 as well as a decrease of H3K9me2 at LSD2-binding sites and a consequent dysregulation of target gene transcription. Furthermore, characterization of the LSD2 complex reveals that LSD2 forms active complexes with euchromatic histone methyltransferases G9a and NSD3 as well as cellular factors involved in transcription elongation. These data provide a possible molecular mechanism linking LSD2 to transcriptional regulation after initiation.

High-throughput β-galactosidase assay for bacterial cell-based reporter systems

Reporting on a Reporter Routine measurement of lacZ expression (β-galactosidase) in bacterial two-hybrid assays is used to determine the relative strengths of various protein-DNA and protein-protei...

Changes in circulating levels of interleukin 6 in burned patients

Interleukin 6 (IL-6) levels in serial serum samples of 10 burned patients were analyzed. The total body surface areas (TBSA) of the burn injury varied from 30 to 85%. Among these 10 patients, five recovered and the other five, who were septic, expired. A significant difference in serum IL-6 values on admission (5-13 h postburn) was found (p < 0.05) between patients who survived or died from burn injury as analyzed by the Wilcoxons rank sum test. In addition, a significant difference in serum IL-6 on admission was also found (p < 0.05) between patients with TBSA of greater or less than 50%. Afterwards, an initial peak serum IL-6 response was detected within 4 days postburn. Significant differences in the peak serum IL-6 levels were not found between patients with TBSA of greater or less than 50% and patients who survived or expired from burn injury. In the survivors, serum IL-6 remained low, while IL-6 increased markedly starting at about one to two weeks postburn in four of the five nonsurvivors with proven sepsis. Except for the patient who expired 42 days postburn, the maximum serum IL-6 values of the other four nonsurvivors were all greater than those of the five survivors from burn injury. Significant correlation (p < 0.05) relating the change in serum IL-6 and body temperature was observed in only two (one survivor and one nonsurvivor) of the ten patients. Changes in serum IL-6 were also compared with changes in circulating TNF-alpha and IL-8 determined previously. A similar pattern in the dynamic changes of circulating TNF-alpha, IL-8 and IL-6 was observed in the individual burned patient. An increase in serum levels of all three cytokines was detected postburn. Serum levels of three cytokines were significantly higher in the septic patients, who all died. It was considered that all three cytokines analyzed may play a significant role in the pathophysiology of sepsis in burned patients.

Deficient transforming growth factor β and interleukin-10 responses contribute to the septic death of burned patients

In order to understand the roles of pro-inflammatory and anti-inflammatory cytokines in burn injury and sepsis post-burn, serial changes in serum levels of transforming growth factor beta-1 (TGF-beta-1) were determined and compared to those of IL-6 and IL-10 in 15 burned patients. Among these 15 patients, 8 recovered without sepsis. The other seven, who were septic, expired. Our results showed that an initial peak serum TGF-beta-1 response was detected within 1 day post-burn. Peak serum IL-6 and IL-10 responses were also detected within 4 days after the burn injury of these patients. Significant differences in peak serum IL-6, IL-10 and TGF-beta-1 levels were not found between patients with total body surface area (TBSA) of greater or less than 50% and between patients who survived or expired from burn injury. Afterwards, levels of circulating IL-6 and IL-10 remained low in the survivors. However, a second peak response in serum TGF-beta-1 levels was observed in all burned patients analyzed. The second peak serum TGF-beta-1 levels post-burn of the eight survivors and the seven non-survivors were from 28,542 to 76,554 pg/ml (a mean value of 51,256+/-14,264 pg/ml) and from 8616 to 40,851 pg/ml (a mean value of 24,079+/-10,399 pg/ml), respectively. A significant difference (P<0.01) in mean values of the second peak TGF-beta-1 responses between groups of survivors and non-survivors was detected. Levels of circulating IL-6 in the septic non-surviving patients showed a tendency to increase 1-2 weeks post-burn and reached high levels before the expiration of these patients. After an initial peak response, the serum IL-10 level remained low in one of the seven non-survivors, while it increased in the other six non-survivors. However, marked increases in circulating IL-10 levels were observed only just before the death of these non-survivors. In conclusion, an initial increase in serum levels of IL-6, IL-10 and TGF-beta-1 was detected post-burn. A marked increase in serum levels of IL-6 before death suggests its role in the pathophysiology of sepsis in burned patients. In addition, a low secondary TGF-beta-1 response and a lack and/or delay in the increase of circulating IL-10 in the non-survivors may all contribute to the pathophysiology of septic death in burned patients.

LSD2/KDM1B and its cofactor NPAC/GLYR1 endow a structural and molecular model for regulation of H3K4 demethylation

Dynamic regulation of histone methylation represents a fundamental epigenetic mechanism underlying eukaryotic gene regulation, yet little is known about how the catalytic activities of histone demethylases are regulated. Here, we identify and characterize NPAC/GLYR1 as an LSD2/KDM1b-specific cofactor that stimulates H3K4me1 and H3K4me2 demethylation. We determine the crystal structures of LSD2 alone and LSD2 in complex with the NPAC linker region in the absence or presence of histone H3 peptide, at resolutions of 2.9, 2.0, and 2.25 Å, respectively. These crystal structures and further biochemical characterization define a dodecapeptide of NPAC (residues 214-225) as the minimal functional unit for its cofactor activity and provide structural determinants and a molecular mechanism underlying the intrinsic cofactor activity of NPAC in stimulating LSD2-catalyzed H3K4 demethylation. Thus, these findings establish a model for how a cofactor directly regulates histone demethylation and will have a significant impact on our understanding of catalytic-activity-based epigenetic regulation.

Changes in serum tumour necrosis factor-α in burned patients

Dynamic tumour necrosis factor-α (TNF-α) changes in serial serum samples of 10 burned patients were analysed in this study. The total body surface areas (TBSA) of the burn injury were from 30 to 85 per cent. Among these 10 patients, five recovered and another five died with proved sepsis. On admission which was about 5–13 h postburn, eight of the 10 patients showed their serum TNF-α levels to be higher than the mean serum TNF-α value of five healthy laboratory personnel. Furthermore, an initial peak serum TNF-α response which could be detected within 2.5 days after burn injury has also been observed. However, significant differences in both the serum TNF-α values on admission, as well as the first peak serum TNF-α levels, were not found (P > 0.05) between patients with TBSA of greater or less than 50 per cent and patients who survived or died from burn injury. In the survivors, serum TNF-α stayed at low levels, while it increased markedly in four of the five non-survivors with proven sepsis starting at about 1 week postburn. A significant difference in the maximum serum TNF-α levels (P < 0.05) was detected between patients who recovered and died from the thermal injury. In conclusion, great increases in serum TNF-α levels have been detected in burned patients with the occurrence of bacterial infection postburn. It is suggested that strategies for the inhibition of TNF-α production or in the neutralization of TNF-α activity should also be considered in the better treatment of burned patients.

A Combined Yeast/Bacteria Two-hybrid System Development and Evaluation

Two-hybrid screening is a standard method used to identify and characterize protein-protein interactions and has become an integral component of many proteomic investigations. The two-hybrid system was initially developed using yeast as a host organism. However, bacterial two-hybrid systems have also become common laboratory tools and are preferred in some circumstances, although yeast and bacterial two-hybrid systems have never been directly compared. We describe here the development of a unified yeast and bacterial two-hybrid system in which a single bait expression plasmid is used in both organismal milieus. We use a series of leucine zipper fusion proteins of known affinities to compare interaction detection using both systems. Although both two-hybrid systems detected interactions within a comparable range of interaction affinities, each demonstrated unique advantages. The yeast system produced quantitative readout over a greater dynamic range than that observed with bacteria. However, the phenomenon of “autoactivation” by baits was less of a problem in the bacterial system than in the yeast. Both systems identified physiological interactors for a library screen with a cI-Ras test bait; however, non-identical interactors were obtained in yeast and bacterial screens. The ability to rapidly shift between yeast and bacterial systems provided by these new reagents should provide a marked advantage for two-hybrid investigations. In addition, the modified expression vectors we describe in this report should be useful for any application requiring facile expression of a protein of interest in both yeast and bacteria.