The Role of VIN3-LIKE Genes in Environmentally Induced Epigenetic Regulation of Flowering

Given their sessile nature, it is critical for the survival of plants to adapt to their environment. Accordingly, plants have evolved the ability to sense seasonal changes to govern developmental fates such as the floral transition. Temperature and day length are among the seasonal cues that plants sense. We recently reported that VIN3-LIKE 1 (VIL1) is involved in mediating the flowering response to both cold and day length via regulation of two related genes, FLOWERING LOCUS C (FLC) and FLOWERING LOCUS M (FLM), respectively.Key Words: flowering, vernalization, photoperiod, chromatin, histone, gene expressionVernalization renders plants competent to flower after exposure to the prolonged cold of winter.^1,2 Arabidopsis exhibits facultative responses to both vernalization and photoperiod to initiate the floral transition. The facultative nature of the responses makes Arabidopsis a tractable genetic system to study these aspects of flowering time control.In Arabidopsis, vernalization creates competence to flower via silencing of the potent floral repressor, FLC, in a mitotically stable manner.^3,4 Thus, the vernalization response is an environmentally induced epigenetic switch in that exposure to cold permanently affects the plants'' developmental program. This epigenetic switch is associated with increased levels of FLC chromatin methylation on Histone H3 Lys 9 and Lys 27.^5,6 VERNALIZATION INSENSITIVE 3 (VIN3) plays an essential role in this switch since no modifications to FLC chromatin occur in vin3 mutants.⁵ Furthermore, the levels of expression of VIN3 mRNA are tightly correlated with the degree of the vernalization response.⁵ VIN3 encodes Plant HomeoDomain (PHD) finger-containing protein. PHD finger-containing proteins are often associated with protein complexes that are involved in chromatin remodeling.⁷We performed a yeast two-hybrid screen to identify potential protein partners of VIN3. VIN3-LIKE 1 (VIL1) was identified by this screen.⁸ VIL1 encodes a PHD finger-containing protein that is related to VIN3. As expected for proteins that are associated with VIN3, plants containing loss-of-function alleles of VIL1 do not respond to vernalization. Furthermore, no vernalization-mediated histone modifications occur at FLC in vil1 mutants similar to the situation in vin3 mutants. Thus, by yeast two hybrid and genetic analysis, VIL1 is a bona fide VIN3 partner that is required for vernalization-mediated histone modifications at FLC chromatin. Unlike VIN3, the expression of VIL1 does not change over the course of cold exposure. Rather, VIL1 mRNA levels are affected by photoperiod. VIL1 expression is significantly increased in non-inductive photoperiods (short days; SD). Consistent with this expression pattern, vil1 mutants in the Columbia accession exhibit a SD-specific late-flowering phenotype. Furthermore, VIL1 is required for attenuating expression of FLOWERING LOCUS M, a FLC-related gene, in a SD-specific manner. It is possible that the attenuation of FLM by VIL1 has a role in creating the facultative nature of photoperiod response in Arabidopsis since vil1 mutants tend towards an obligate photoperiod response (i.e., vil1 mutants often fail to flower in SD).In Arabidopsis, there are four VIN3-related genes, which we named as VIL1 ∼ VIL4,⁸ and which have also been called VRN5 and VEL1 ∼ VEL3.⁹ The C-terminal domain is highly conserved among these genes and was named the VIN3-Interacting Domain (VID) since it is required for protein-protein interaction between VIN3 and VIL1. The effect of cold on the expression patterns of VIN3-related genes varies. For example, VIL2 and VIL3 are induced specifically by vernalizing cold exposures whereas others such as VIL1 are, for the most part, constitutively expressed. It will be interesting to determine the functions of the remaining VIL genes.FLC is the main target for vernalization in Arabidopsis. Interestingly, FLC orthologs have not been found in vernalization-responsive varieties of cereals. However, in wheat, VRN2 appears to have a role equivalent to that of FLC in Arabidopsis.¹⁰ VRN2 encodes a ZCCT type zinc-finger protein that does not have a homolog in the Arabidopsis genome. In diploid wheat, down regulation of VRN2 is correlated with the vernalization response.¹¹ Interestingly, wheat contains three VIN3-LIKE (VIL) genes, TmVIL1, TmVIL2 and TmVIL3.¹² Furthermore, TmVIL1 is up-regulated by vernalization.¹² However, whether TmVIL1 has a direct role in the vernalization-mediated repression of VRN2 in wheat has not yet been addressed. Similar to VIL1, TmVIL3 shows elevated level of expression in SD. Furthermore, VRN2 is downregulated in SD;^13,14 thus there is a correlation between the induction of TmVIL genes and the downregulation of the floral repressor VRN2 similar to the VIN3/FLC and VIL1/FLM relationships (Fig. 1). Perhaps VIN3-related genes have similar roles both in Arabidopsis and in temperate wheat, but act on different target genes, possibly as a result of convergent evolution. Interestingly, the wheat gene TmVRN3 is homologous to FLOWERING LOCUS T (FT) of Arabidopsis, and TmVRN3 is repressed by TmVRN2 as FT is repressed by FLC,¹⁵ suggesting another similarity in the regulation of flowering time between Arabidopsis and temperate wheat (Fig. 1).Open in a separate windowFigure 1Proposed relationship of VIN3 family genes to the regulatory network controlling flowering time in response to environmental cues in Arabidopsis and diploid wheat (adapted from ref. ¹⁶).Although the PHD finger can be found in various eukaryotes, the VID is unique to plants. It is also noteworthy that VIN3-related genes can be found in various plant species, including rice, which does not have a vernalization response. It will be interesting to address whether the VIN3-related genes from various plant species are more broadly involved in relaying environmental signals to developmental programs.     

Monocyte cholesterol homeostasis correlates with the presence of detergent resistant membrane microdomains.

BACKGROUND: Lipid membrane microdomains are involved in the regulation of biological functions of monocyte membrane proteins. These microdomains show a relative resistance to non-ionic detergents providing an easy analytical tool to study them. METHODS: Here, we applied a rapid detergent-based flow cytometric assay to investigate microdomain association of proteins on monocytes from whole blood samples. The association of known surface antigens with detergent resistant fraction of membranes (DRMs) was compared using monocytes from healthy blood donors, patients with genetic disorders affecting cellular cholesterol traffic and patients with systemic inflammatory response. RESULTS: All investigated surface antigens of Niemann-Pick type C (NPC)-mutant monocytes with impaired cholesterol influx and defective late endosome cholesterol trafficking, presented a strongly increased DRM-association. Though, membrane antigens of ATP binding cassette transporter A1 (ABCA1)-mutant monocytes with impaired cholesterol efflux did not show alterations in DRM-association. Differential CD14-dependent receptor clustering within microdomains was also investigated in response to in vivo lipopolysaccharide (LPS) and/or atherogenic lipoprotein activation. Increased DRM-association of the GPI-anchored proteins CD14, CD55, the Fcgamma receptor CD64, the scavenger receptors CD36, CD91 and CD163, the integrin CD11a, and complement receptor 3 complex CD11b/CD18 were observed from patients with systemic inflammatory response syndrome (SIRS)/sepsis or coronary artery disease (CAD)/myocardial infarction. Interestingly, the tetraspanin CD81 presented increased DRM-association in SIRS/sepsis patients, but not in CAD patients. Moreover, the pentaspanin CD47 and the Fcgamma RIII CD16 showed an increased DRM partition in CAD patients but disassembled from DRMs in SIRS/sepsis patients. CONCLUSIONS: Our results demonstrate that flow cytometric analysis of short time in situ detergent extraction provides a powerful tool for rapid screening of blood monocyte DRMs to preselect patients with potential raft/microdomain abnormalities for more detailed analysis.     

The TCR repertoire of an immunodominant CD8+ T lymphocyte population

The TCR repertoire of an epitope-specific CD8(+) T cell population remains poorly characterized. To determine the breadth of the TCR repertoire of a CD8(+) T cell population that recognizes a dominant epitope of the AIDS virus, the CD8(+) T cells recognizing the tetrameric Mamu-A*01/p11C(,CM) complex were isolated from simian immunodeficiency virus (SIV)-infected Mamu-A*01(+) rhesus monkeys. This CD8(+) T cell population exhibited selected usage of TCR V beta families and complementarity-determining region 3 (CDR3) segments. Although the epitope-specific CD8(+) T cell response was clearly polyclonal, a dominance of selected V beta(+) cell subpopulations and clones was seen in the TCR repertoire. Interestingly, some of the selected V beta(+) cell subpopulations and clones maintained their dominance in the TCR repertoire over time after infection with SIV of macaques. Other V beta(+) cell subpopulations declined over time in their relative representation and were replaced by newly evolving clones that became dominant. The present study provides molecular evidence indicating that the TCR repertoire shaped by a single viral epitope is dominated at any point in time by selected V beta(+) cell subpopulations and clones and suggests that dominant V beta(+) cell subpopulations and clones can either be stable or evolve during a chronic infection.     

ApoE-containing high density lipoproteins and phospholipid transfer protein activity increase in patients with a systemic inflammatory response

High density lipoproteins (HDL) mediate reverse cholesterol transport as well as the clearance of oxidation products or inflammatory mediators, thereby contributing to tissue integrity. The decrease in HDL in inflammation has been attributed to decreased lecithin:cholesterol acyltransferase activity, whereas the role of phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein has not been analyzed in detail. We have studied the activities of HDL-modifying proteins and the heterogeneity of HDL in healthy control subjects and three groups of postsurgery patients: no bacterial infection (group 1), bacterial focus and systemic inflammatory response (group 2), and severe sepsis (group 3). For all patients, a decrease in total HDL could be demonstrated, with a loss of mainly large, apolipoprotein A-I (apoA-I) HDL particles, an almost total loss of apoC-I, and an increase in apoE HDL (200-500 kDa), which did not contain significant amounts of apoA-I, apoA-II, or apoC-I. PLTP activity was increased in patients of groups 2 and 3, paralleled by a redistribution of PLTP into a population of small (120- to 200-kDa) particles, probably representing PLTP homodimers or lipid-complexed PLTP.In summary, the increase in apoE HDL and PLTP activity may improve the delivery of energy substrates and phospholipids to tissues that must maintain cellular membrane homeostasis under conditions of inflammatory stress.     

Reduction of simian-human immunodeficiency virus 89.6P viremia in rhesus monkeys by recombinant modified vaccinia virus Ankara vaccination

Since cytotoxic T lymphocytes (CTLs) are critical for controlling human immunodeficiency virus type 1 (HIV-1) replication in infected individuals, candidate HIV-1 vaccines should elicit virus-specific CTL responses. In this report, we study the immune responses elicited in rhesus monkeys by a recombinant poxvirus vaccine and the degree of protection afforded against a pathogenic simian-human immunodeficiency virus SHIV-89.6P challenge. Immunization with recombinant modified vaccinia virus Ankara (MVA) vectors expressing SIVmac239 gag-pol and HIV-1 89.6 env elicited potent Gag-specific CTL responses but no detectable SHIV-specific neutralizing antibody (NAb) responses. Following intravenous SHIV-89.6P challenge, sham-vaccinated monkeys developed low-frequency CTL responses, low-titer NAb responses, rapid loss of CD4+ T lymphocytes, high-setpoint viral RNA levels, and significant clinical disease progression and death in half of the animals by day 168 postchallenge. In contrast, the recombinant MVA-vaccinated monkeys demonstrated high-frequency secondary CTL responses, high-titer secondary SHIV-89.6-specific NAb responses, rapid emergence of SHIV-89.6P-specific NAb responses, partial preservation of CD4+ T lymphocytes, reduced setpoint viral RNA levels, and no evidence of clinical disease or mortality by day 168 postchallenge. There was a statistically significant correlation between levels of vaccine-elicited CTL responses prior to challenge and the control of viremia following challenge. These results demonstrate that immune responses elicited by live recombinant vectors, although unable to provide sterilizing immunity, can control viremia and prevent disease progression following a highly pathogenic AIDS virus challenge.     

Sequence, purification, and cloning of an intracellular serine protease, quiescent cell proline dipeptidase

We recently observed that specific inhibitors of post-proline cleaving aminodipeptidases cause apoptosis in quiescent lymphocytes in a process independent of CD26/dipeptidyl peptidase IV. These results led to the isolation and cloning of a new protease that we have termed quiescent cell proline dipeptidase (QPP). QPP activity was purified from CD26(-) Jurkat T cells. The protein was identified by labeling with [(3)H]diisopropylfluorophosphate and subjected to tryptic digestion and partial amino acid sequencing. The peptide sequences were used to identify expressed sequence tag clones. The cDNA of QPP contains an open reading frame of 1476 base pairs, coding for a protein of 492 amino acids. The amino acid sequence of QPP reveals similarity with prolylcarboxypeptidase. The putative active site residues serine, aspartic acid, and histidine of QPP show an ordering of the catalytic triad similar to that seen in the post-proline cleaving exopeptidases prolylcarboxypeptidase and CD26/dipeptidyl peptidase IV. The post-proline cleaving activity of QPP has an unusually broad pH range in that it is able to cleave substrate molecules at acidic pH as well as at neutral pH. QPP has also been detected in nonlymphocytic cell lines, indicating that this enzyme activity may play an important role in other tissues as well.     

Endoplasmic reticulum-associated degradation: exceptions to the rule

Quality control mechanisms in the endoplasmic reticulum (ER) ensure that misfolded proteins are recognized and targeted for degradation. According to the current view of ER-associated degradation (ERAD), the degradation does not occur in the ER itself but requires the retrotranslocation of the proteins to the cytosol where they are degraded by proteasomes. Although this model appears to be valid for many different proteins a number of exceptions from this rule suggest that additional proteasome-independent ERAD pathways may exist. In this review, we will summarize what is known about these alternative ERAD pathways.