Prognosis of Patients With Stages I and II Non-Small Cell Lung Cancer With Nodal Micrometastases.

BACKGROUND: This retrospective study aimed to determine the prognostic significance of immunohistochemically detected occult micrometastases (OM) in mediastinal lymph nodes (LNs) obtained during transcervical extended mediastinal lymphadenectomy of stages I and II non-small cell small cancer (NSCLC) patients before complete surgical resection. METHODS: From January 2007 to June 2011, 75 patients with pathologic stage I NSCLC and 73 patients with pathologic stage II NSCLC underwent transcervical extended mediastinal lymphadenectomy and subsequent radical pulmonary resection. During transcervical extended mediastinal lymphadenectomy, 4,810 mediastinal LNs resected and determined as metastases-free by hematoxylin and eosin staining were immunohistochemically labelled with anticytokeratin and BerEp4 antibodies to detect OM. RESULTS: OM were detected in 9 mediastinal LNs of 7 stage I (9.3%) and in 10 mediastinal LNs of 7 stage II (9.6%) NSCLC patients. Patients with mediastinal LN OM had reduced 5-year disease-free and overall survival (21.4%) compared with stage I (61.8%, p < 0.001) and stage II (47.0%, p < 0.05) patients. Multivariable analysis showed the presence of OM was a significant negative prognostic factor for 5-year overall and disease-free survival rates. CONCLUSIONS: The presence of OM in the mediastinal LNs was associated with decreased total and disease-free survival rates in stages I and II NSCLC patients. Immunohistochemical staining of mediastinal LNs obtained preoperatively improved the accuracy of staging and allowed for the identification of patients with a poorer prognosis.

Distinct and overlapping DNMT1 interactions with multiple transcription factors in erythroid cells: Evidence for co-repressor functions.

DNMT1 is the maintenance DNA methyltransferase shown to be essential for embryonic development and cellular growth and differentiation in many somatic tissues in mammals. Increasing evidence has also suggested a role for DNMT1 in repressing gene expression through interactions with specific transcription factors. Previously, we identified DNMT1 as an interacting partner of the TR2/TR4 nuclear receptor heterodimer in erythroid cells, implicated in the developmental silencing of fetal ß-type globin genes in the adult stage of human erythropoiesis. Here, we extended this work by using a biotinylation tagging approach to characterize DNMT1 protein complexes in mouse erythroleukemic cells. We identified novel DNMT1 interactions with several hematopoietic transcription factors with essential roles in erythroid differentiation, including GATA1, GFI-1b and FOG-1. We provide evidence for DNMT1 forming distinct protein subcomplexes with specific transcription factors and propose the existence of a "core" DNMT1 complex with the transcription factors ZBP-89 and ZNF143, which is also present in non-hematopoietic cells. Furthermore, we identified the short (17a.a.) PCNA Binding Domain (PBD) located near the N-terminus of DNMT1 as being necessary for mediating interactions with the transcription factors described herein. Lastly, we provide evidence for DNMT1 serving as a co-repressor of ZBP-89 and GATA1 acting through upstream regulatory elements of the PU.1 and GATA1 gene loci.

Biased, non-equivalent gene-proximal and -distal binding motifs of orphan nuclear receptor TR4 in primary human erythroid cells.

We previously reported that TR2 and TR4 orphan nuclear receptors bind to direct repeat (DR) elements in the ε- and γ-globin promoters, and act as molecular anchors for the recruitment of epigenetic corepressors of the multifaceted DRED complex, thereby leading to ε- and γ-globin transcriptional repression during definitive erythropoiesis. Other than the ε- and γ-globin and the GATA1 genes, TR4-regulated target genes in human erythroid cells remain unknown. Here, we identified TR4 binding sites genome-wide using chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) as human primary CD34(+) hematopoietic progenitors differentiated progressively to late erythroid precursors. We also performed whole transcriptome analyses by RNA-seq to identify TR4 downstream targets after lentiviral-mediated TR4 shRNA knockdown in erythroid cells. Analyses from combined ChIP-seq and RNA-seq datasets indicate that DR1 motifs are more prevalent in the proximal promoters of TR4 direct target genes, which are involved in basic biological functions (e.g., mRNA processing, ribosomal assembly, RNA splicing and primary metabolic processes). In contrast, other non-DR1 repeat motifs (DR4, ER6 and IR1) are more prevalent at gene-distal TR4 binding sites. Of these, approximately 50% are also marked with epigenetic chromatin signatures (such as P300, H3K27ac, H3K4me1 and H3K27me3) associated with enhancer function. Thus, we hypothesize that TR4 regulates gene transcription via gene-proximal DR1 sites as TR4/TR2 heterodimers, while it can associate with novel nuclear receptor partners (such as RXR) to bind to distant non-DR1 consensus sites. In summary, this study reveals that the TR4 regulatory network is far more complex than previously appreciated and that TR4 regulates basic, essential biological processes during the terminal differentiation of human erythroid cells.

Nuclear receptors TR2 and TR4 recruit multiple epigenetic transcriptional corepressors that associate specifically with the embryonic ß-type globin promoters in differentiated adult erythroid cells.

Nuclear receptors TR2 and TR4 (TR2/TR4) were previously shown to bind in vitro to direct repeat elements in the mouse and human embryonic and fetal ß-type globin gene promoters and to play critical roles in the silencing of these genes. By chromatin immunoprecipitation (ChIP) we show that, in adult erythroid cells, TR2/TR4 bind to the embryonic ß-type globin promoters but not to the adult ß-globin promoter. We purified protein complexes containing biotin-tagged TR2/TR4 from adult erythroid cells and identified DNMT1, NuRD, and LSD1/CoREST repressor complexes, as well as HDAC3 and TIF1ß, all known to confer epigenetic gene silencing, as potential corepressors of TR2/TR4. Coimmunoprecipitation assays of endogenous abundance proteins indicated that TR2/TR4 complexes consist of at least four distinct molecular species. In ChIP assays we found that, in undifferentiated murine adult erythroid cells, many of these corepressors associate with both the embryonic and the adult ß-type globin promoters but, upon terminal differentiation, they specifically dissociate only from the adult ß-globin promoter concomitant with its activation but remain bound to the silenced embryonic globin gene promoters. These data suggest that TR2/TR4 recruit an array of transcriptional corepressors to elicit adult stage-specific silencing of the embryonic ß-type globin genes through coordinated epigenetic chromatin modifications.

Optimal use of tandem biotin and V5 tags in ChIP assays.

BACKGROUND: Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies. RESULTS: Using the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking. CONCLUSION: The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes.

Isolation of transcription factor complexes by in vivo biotinylation tagging and direct binding to streptavidin beads.

Efficient tagging methodologies are an integral aspect of protein complex characterization by proteomic approaches. Owing to the very high affinity of biotin for avidin and streptavidin, biotinylation tagging offers an attractive approach for the efficient purification of protein complexes. The very high affinity of the biotin/(strept)avidin system also offers the potential for the single-step capture of lower abundance protein complexes, such as transcription factor complexes. The identification of short peptide tags that are efficiently biotinylated by the bacterial BirA biotin ligase led to an approach for the single-step purification of transcription factor complexes by specific in vivo biotinylation tagging. A short sequence tag fused N-terminally to the transcription factor of interest is very efficiently biotinylated by BirA coexpressed in the same cells, as was demonstrated by the tagging of the essential hematopoietic transcription factor GATA-1. The direct binding to streptavidin of biotinylated GATA-1 in nuclear extracts resulted in the single-step capture of the tagged factor and associated proteins, which were eluted and identified by mass spectrometry. This led to the characterization of several distinct GATA-1 complexes with other transcription factors and chromatin remodeling cofactors, which are involved in activation and repression of gene targets. Thus, BirA-mediated tagging is an efficient approach for the direct capture and characterization of transcription factor complexes.

Isolation and characterization of hematopoietic transcription factor complexes by in vivo biotinylation tagging and mass spectrometry.

We have described the application of a simple biotinylation tagging approach for the direct purification of tagged transcription factor complexes, based on the use of artificial short peptide tags that are specifically and efficiently biotinylated by the bacterial BirA biotin ligase, which is co-expressed in cells with the tagged factor. We used this approach to initially characterize complexes formed by the hematopoietic transcription factor GATA-1 in erythroid cells. GATA-1 is essential for the erythroid differentiation, its functions encompassing upregulation of erythroid genes, repression of alternative transcription programs, and suppression of cell proliferation. However, it was not clear how all of these GATA-1 functions are mediated. Our work describes, for the first time, distinct GATA-1 interactions with the essential hematopoietic factor Gfi-1b, the repressive MeCP1 complex, and the chromatin remodeling ACF/WCRF complex, in addition to the known GATA-1/FOG-1 and GATA-1/TAL-1 complexes. We also provide evidence that distinct GATA-1 complexes are associated with specific GATA-1 functions in erythroid differentiation, for example, GATA-1/Gfi-1b with the suppression of cell proliferation and GATA-1/FOG-1/MeCP1 with the repression of other hematopoietic transcription programs. We next applied the biotinylation tag to Ldb-1, a known partner of GATA-1, and characterized a number of novel interaction partners that are essential in erythroid development, in particular, Eto-2, Lmo4, and CdK9. Last, we are in the process of applying the same technology to characterize the factors that are bound to the suppressed gamma-globin promoter in vivo.

GATA-1 forms distinct activating and repressive complexes in erythroid cells.

GATA-1 is essential for the generation of the erythroid, megakaryocytic, eosinophilic and mast cell lineages. It acts as an activator and repressor of different target genes, for example, in erythroid cells it represses cell proliferation and early hematopoietic genes while activating erythroid genes, yet it is not clear how both of these functions are mediated. Using a biotinylation tagging/proteomics approach in erythroid cells, we describe distinct GATA-1 interactions with the essential hematopoietic factor Gfi-1b, the repressive MeCP1 complex and the chromatin remodeling ACF/WCRF complex, in addition to the known GATA-1/FOG-1 and GATA-1/TAL-1 complexes. Importantly, we show that FOG-1 mediates GATA-1 interactions with the MeCP1 complex, thus providing an explanation for the overlapping functions of these two factors in erythropoiesis. We also show that subsets of GATA-1 gene targets are bound in vivo by distinct complexes, thus linking specific GATA-1 partners to distinct aspects of its functions. Based on these findings, we suggest a model for the different roles of GATA-1 in erythroid differentiation.

A link between Helicobacter pylori and/or Chlamydia spp. infections and atherosclerosis.

Antibodies to Helicobacter pylori, Chlamydia spp. and Mycobacterium bovis were determined in patients with coronary heart disease, H. pylori-related dyspepsia, and tuberculosis, and healthy controls. Enzyme-linked immunosorbent assay was conducted with a glycine extract and CagA protein of H. pylori, chlamydial lipopolysaccharide and mycobacterial heat shock protein Hsp65. The prevalence of anti-glycine extract IgG in coronary heart disease patients was higher than in the tuberculosis group and controls, and the same as in dyspeptic patients. Anti-chlamydial IgG were more prevalent in the coronary heart disease group than in healthy subjects. There was no difference in the prevalence of anti-CagA IgG in the coronary heart disease group and controls or anti-Hsp65 IgG in the patients with coronary heart disease, dyspepsia, tuberculosis, and controls. Anti-glycine extract IgA (like anti-glycine extract IgG) were more prevalent in the coronary heart disease group than in the healthy group. The highest anti-glycine extract IgG/IgA and anti-chlamydial IgG titers were more frequent in coronary heart disease patients as compared with controls. Infections with H. pylori and Chlamydia spp. and enhanced production of antibodies to these pathogens may predispose to human atherosclerosis.