The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

/pdf/initial-sequencing-and-analysis-of-the-human-genome-5dapk1rsx1.pdf

Initial sequencing and analysis of the human genome.

The character of a cell is defined by its constituent proteins, which are the result of specific patterns of gene expression. Crucial determinants of gene expression patterns are DNA-binding transcription factors that choose genes for transcriptional activation or repression by recognizing the sequence of DNA bases in their promoter regions. Interaction of these factors with their cognate sequences triggers a chain of events, often involving changes in the structure of chromatin, that leads to the assembly of an active transcription complex (e.g., Cosma et al. 1999). But the types of transcription factors present in a cell are not alone sufficient to define its spectrum of gene activity, as the transcriptional potential of a genome can become restricted in a stable manner during development. The constraints imposed by developmental history probably account for the very low efficiency of cloning animals from the nuclei of differentiated cells (Rideout et al. 2001; Wakayama and Yanagimachi 2001). A “transcription factors only” model would predict that the gene expression pattern of a differentiated nucleus would be completely reversible upon exposure to a new spectrum of factors. Although many aspects of expression can be reprogrammed in this way (Gurdon 1999), some marks of differentiation are evidently so stable that immersion in an alien cytoplasm cannot erase the memory. The genomic sequence of a differentiated cell is thought to be identical in most cases to that of the zygote from which it is descended (mammalian B and T cells being an obvious exception). This means that the marks of developmental history are unlikely to be caused by widespread somatic mutation. Processes less irrevocable than mutation fall under the umbrella term “epigenetic” mechanisms. A current definition of epigenetics is: “The study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence” (Russo et al. 1996). There are two epigenetic systems that affect animal development and fulfill the criterion of heritability: DNA methylation and the Polycomb-trithorax group (Pc-G/trx) protein complexes. (Histone modification has some attributes of an epigenetic process, but the issue of heritability has yet to be resolved.) This review concerns DNA methylation, focusing on the generation, inheritance, and biological significance of genomic methylation patterns in the development of mammals. Data will be discussed favoring the notion that DNA methylation may only affect genes that are already silenced by other mechanisms in the embryo. Embryonic transcription, on the other hand, may cause the exclusion of the DNA methylation machinery. The heritability of methylation states and the secondary nature of the decision to invite or exclude methylation support the idea that DNA methylation is adapted for a specific cellular memory function in development. Indeed, the possibility will be discussed that DNA methylation and Pc-G/trx may represent alternative systems of epigenetic memory that have been interchanged over evolutionary time. Animal DNA methylation has been the subject of several recent reviews (Bird and Wolffe 1999; Bestor 2000; Hsieh 2000; Costello and Plass 2001; Jones and Takai 2001). For recent reviews of plant and fungal DNA methylation, see Finnegan et al. (2000), Martienssen and Colot (2001), and Matzke et al. (2001).

/pdf/dna-methylation-patterns-and-epigenetic-memory-2xnqjp4ije.pdf

DNA methylation patterns and epigenetic memory

Monocytes originate from progenitors in the bone marrow and traffic via the bloodstream to peripheral tissues. During both homeostasis and inflammation, circulating monocytes leave the bloodstream and migrate into tissues where, following conditioning by local growth factors, pro-inflammatory cytokines and microbial products, they differentiate into macrophage or dendritic cell populations. Recruitment of monocytes is essential for effective control and clearance of viral, bacterial, fungal and protozoal infections, but recruited monocytes also contribute to the pathogenesis of inflammatory and degenerative diseases. The mechanisms that control monocyte trafficking under homeostatic, infectious and inflammatory conditions are being unravelled and are the focus of this Review.

/pdf/monocyte-recruitment-during-infection-and-inflammation-2qbag1l4e4.pdf

Monocyte recruitment during infection and inflammation

All eukaryotic cells have an extensive membranous labyrinth network of branching tubules and flattened sacs called the endoplasmic reticulum (ER). Approximately one-third of all cellular proteins are translocated into the lumen of the ER where post-translational modification, folding, and oligomerization occurs. The ER provides a unique oxidizing compartment for the folding of membrane and secretory proteins that are destined to the cell surface, as well as for proteins destined to other intracellular organelles, such as lysosomes and the Golgi compartment. Numerous cellular proteins reside within the ER through a mechanism that requires their continuous vesicle-mediated retrieval from post-ER compartments within the early secretory pathway. These ERresident proteins are chaperones and catalysts of protein folding that form a matrix on which newly synthesized proteins attain their final conformation. The ER is also the site of synthesis of cellular lipids and sterols. In addition, the ER is the major signal-transducing organelle within the cell that continuously responds to environmental cues to release calcium. The ER is exquisitely sensitive to alterations in homeostasis, where, upon a variety of different stimuli, signals are transduced from the ER to the cytoplasm and the nucleus to eventually result in adaptation for survival or induction of apoptosis. The immediate response occurs at the translational apparatus, whereas changes in gene expression promote long-term adaptation or apoptotic cell death. Recent evidence supports findings that these signaling pathways influence pathogenesis associated with viral infection and genetic disease. The purpose of this review is to summarize what is presently known about the diversity of molecular signaling mechanisms that coordinate the complex ER stress response at the translational and transcriptional level in yeast and in higher eukaryotic cells.

/pdf/stress-signaling-from-the-lumen-of-the-endoplasmic-reticulum-50luw2lbp2.pdf

Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls

Type I interferons (IFNs) have diverse effects on innate and adaptive immune cells during infection with viruses, bacteria, parasites and fungi, directly and/or indirectly through the induction of other mediators. Type I IFNs are important for host defence against viruses. However, recently, they have been shown to cause immunopathology in some acute viral infections, such as influenza virus infection. Conversely, they can lead to immunosuppression during chronic viral infections, such as lymphocytic choriomeningitis virus infection. During bacterial infections, low levels of type I IFNs may be required at an early stage, to initiate cell-mediated immune responses. High concentrations of type I IFNs may block B cell responses or lead to the production of immunosuppressive molecules, and such concentrations also reduce the responsiveness of macrophages to activation by IFNγ, as has been shown for infections with Listeria monocytogenes and Mycobacterium tuberculosis. Recent studies in experimental models of tuberculosis have demonstrated that prostaglandin E2 and interleukin-1 inhibit type I IFN expression and its downstream effects, demonstrating that a cross-regulatory network of cytokines operates during infectious diseases to provide protection with minimum damage to the host.

/pdf/type-i-interferons-in-infectious-disease-2xngbxv19g.pdf

Type I interferons in infectious disease.

A novel role for HMGB1 in TLR9-mediated inflammatory responses to CpG-DNA.

CpG-DNA or its synthetic analog CpG-ODN activates innate immunity through Toll-like receptor 9 (TLR9). However, the mechanism of TLR9 activation by CpG-DNA remains elusive. Here we have identified HMGB1 as a CpG-ODN-binding protein. HMGB1 interacts and preassociates with TLR9 in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), and hastens TLR9's redistribution to early endosomes in response to CpG-ODN. CpG-ODN stimulates macrophages and dendritic cells to secrete HMGB1; in turn, extracellular HMGB1 accelerates the delivery of CpG-ODNs to its receptor, leading to a TLR9-dependent augmentation of IL-6, IL-12, and TNFalpha secretion. Loss of HMGB1 leads to a defect in the IL-6, IL-12, TNFalpha, and iNOS response to CpG-ODN. However, lack of intracellular TLR9-associated HMGB1 can be compensated by extracellular HMGB1. Thus, the DNA-binding protein HMGB1 shuttles in and out of immune cells and regulates inflammatory responses to CpG-DNA.

A novel role for HMGB1 in TLR9-mediated inflammatory responses to CpG-DNA Running title: Mechanism of TLR9 activation

The abundance of Alu RNA is transiently increased by heat shock in human cell lines. This effect is specific to Alu repeats among Pol III transcribed genes, since the abundance of 7SL, 7SK, 5S and U6 RNAs is essentially unaffected by heat shock. The rapid induction of Alu expression precedes the heat shock induction of mRNAs for the ubiquitin and HSP 70 heat shock genes. Heat shock mimetics also transiently induce Alu expression indicating that increased Alu expression is a general cell-stress response. Cycloheximide treatment rapidly and transiently increases the abundance of Alu RNA. Again, compared with other genes transcribed by Pol III, this increase is specific to Alu. However, as distinguished from the cell stress response, cycloheximide does not induce expression of HSP 70 and ubiquitin mRNAs. Puromycin also increases Alu expression, suggesting that this response is generally caused by translational inhibition. The response of mammalian SINEs to cell stress and translational inhibition is not limited to SINEs which are Alu homologues. Heat shock and cycloheximide each transiently induce Pol III directed expression of B1 and B2 RNAs in mouse cells and C-element RNA in rabbit cells. Together, these three species exemplify the known SINE composition of placental mammals, suggesting that mammalian SINEs are similarly regulated and may serve a common function.

Cell stress and translational inhibitors transiently increase the abundance of mammalian SINE transcripts

Cell stress, viral infection, and translational inhibition increase the abundance of human Alu RNA, suggesting that the level of these transcripts is sensitive to the translational state of the cell To determine whether Alu RNA functions in translational homeostasis, we investigated its role in the regulation of double-stranded RNA-activated kinase PKR We found that overexpression of Alu RNA by cotransient transfection increased the expression of a reporter construct, which is consistent with an inhibitory effect on PKR Alu RNA formed stable, discrete complexes with PKR in vitro, bound PKR in vivo, and antagonized PKR activation both in vitro and in vivo Alu RNAs produced by either overexpression or exposure of cells to heat shock bound PKR, whereas transiently overexpressed Alu RNA antagonized virus-induced activation of PKR in vivo Cycloheximide treatment of cells decreased PKR activity, coincident with an increase in Alu RNA These observations suggest that the increased levels of Alu RNAs caused by cellular exposure to different stresses regulate protein synthesis by antagonizing PKR activation This provides a functional role for mammalian short interspersed elements, prototypical junk DNA

Potential Alu Function: Regulation of the Activity of Double-Stranded RNA-Activated Kinase PKR

The best-characterized type 1 interferon (IFN) signaling pathway depends on signal transducer and activator of transcription 1 (STAT1) and STAT2. The cytokines can, however, conditionally activate all STATs. Regulation of their access to particular signaling pathways is poorly understood. STAT4 is important for IFN-γ induction, and NK cells are major producers of this cytokine. We report that NK cells have high basal STAT4 levels and sensitivity to type 1 IFN–mediated STAT4 activation for IFN-γ production. Increases in STAT1, driven during viral infection by either type 1 IFN or IFN-γ, are associated with decreased STAT4 access. Both STAT1 and STAT2 are important for antiviral defense, but STAT1 has a unique role in protecting against sustained NK cell IFN-γ production and resulting disease. The regulation occurs with an NK cell type 1 IFN receptor switch from a STAT4 to a STAT1 association. Thus, a fundamental characteristic of NK cells is high STAT4 bound to the type 1 IFN receptor. The conditions of infection result in STAT1 induction with displacement of STAT4. These studies elucidate the critical role of STAT4 levels in predisposing selection of specific signaling pathways, define the biological importance of regulation within particular cell lineages, and provide mechanistic insights for how this is accomplished in vivo.

https://rupress.org/jem/article-pdf/204/10/2383/1129536/jem_20070401.pdf

Wen-Ming Chu

Papers

A novel role for HMGB1 in TLR9-mediated inflammatory responses to CpG-DNA.

A novel role for HMGB1 in TLR9-mediated inflammatory responses to CpG-DNA Running title: Mechanism of TLR9 activation

Cell stress and translational inhibitors transiently increase the abundance of mammalian SINE transcripts

Potential Alu Function: Regulation of the Activity of Double-Stranded RNA-Activated Kinase PKR

High basal STAT4 balanced by STAT1 induction to control type 1 interferon effects in natural killer cells