Marian Mellén

Bio: Marian Mellén is an academic researcher from Rockefeller University. The author has contributed to research in topics: Gene & Gene expression. The author has an hindex of 5, co-authored 5 publications receiving 1339 citations. Previous affiliations of Marian Mellén include Spanish National Research Council & Howard Hughes Medical Institute.

MeCP2 binds to 5hmc enriched within active genes and accessible chromatin in the nervous system

Abstract: SUMMARY The high level of 5-hydroxymethylcytosine (5hmC) present in neuronal genomes suggests that mechanisms interpreting 5hmC in the CNS may differ from those present in embryonic stem cells. Here, we present quantitative, genome-wide analysis of 5hmC, 5-methylcytosine (5mC), and gene expression in differentiated CNS cell types in vivo. We report that 5hmC is enriched in active genes and that, surprisingly, strong depletion of 5mC is observed over these regions. The contribution of these epigenetic marks to gene expression depends critically on cell type. We identify methyl-CpG-binding protein 2 (MeCP2) as the major 5hmC-binding protein in the brain and demonstrate that MeCP2 binds 5hmC- and 5mC-containing DNA with similar high affinities. The Rett-syndrome-causing mutation R133C preferentially inhibits 5hmC binding. These findings support a model in which 5hmC and MeCP2 constitute a cell-specific epigenetic mechanism for regulation of chromatin structure and gene expression.

5-hydroxymethylcytosine accumulation in postmitotic neurons results in functional demethylation of expressed genes

Abstract: 5-hydroxymethylcytosine (5hmC) occurs at maximal levels in postmitotic neurons, where its accumulation is cell-specific and correlated with gene expression. Here we demonstrate that the distribution of 5hmC in CG and non-CG dinucleotides is distinct and that it reflects the binding specificity and genome occupancy of methylcytosine binding protein 2 (MeCP2). In expressed gene bodies, accumulation of 5hmCG acts in opposition to 5mCG, resulting in "functional" demethylation and diminished MeCP2 binding, thus facilitating transcription. Non-CG hydroxymethylation occurs predominantly in CA dinucleotides (5hmCA) and it accumulates in regions flanking active enhancers. In these domains, oxidation of 5mCA to 5hmCA does not alter MeCP2 binding or expression of adjacent genes. We conclude that the role of 5-hydroxymethylcytosine in postmitotic neurons is to functionally demethylate expressed gene bodies while retaining the role of MeCP2 in chromatin organization.

The autophagic machinery is necessary for removal of cell corpses from the developing retinal neuroepithelium.

Abstract: Autophagy is a homoeostatic process necessary for the clearance of damaged or superfluous proteins and organelles. The recycling of intracellular constituents also provides energy during periods of metabolic stress, thereby contributing to cell viability. In addition, disruption of autophagic machinery interferes with embryonic development in several species, although the underlying cellular processes affected remain unclear. Here, we investigate the role of autophagy during the early stages of chick retina development, when the retinal neuroepithelium proliferates and starts to generate the first neurons, the retinal ganglion cells. These two developmental processes are accompanied by programmed cell death. Upon treatment with the autophagic inhibitor 3-methyladenine, retinas accumulated numerous TdT-mediated dUTP nick-end labelling-positive cells that correlated with a lack of the ‘eat-me’ signal phosphatidylserine (PS). In consequence, neighbouring cells did not engulf apoptotic bodies and they persisted as individual cell corpses, a phenotype that was also observed after blockade of phagocytosis with phospho-L-Serine. Supplying the retinas with methylpyruvate, a cell-permeable substrate for ATP production, restored ATP levels and the presentation of PS at the cell surface. Hence, engulfment and lysosomal degradation of apoptotic bodies were also re-established. Together, these data point to a novel role for the autophagic machinery during the development of the central nervous system.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Abstract: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy

Abstract: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Repertoires of Autophagy in the Pathogenesis of Ocular Diseases

Abstract: Autophagy is an important intracellular degradative process that delivers cytoplasmic proteins to lysosome for degradation. Dysfunction of autophagy is implicated in several human diseases, such as neurodegenerative diseases, infectious diseases, and cancers. Autophagy-related proteins are constitutively expressed in the eye. Increasing studies have revealed that abnormal autophagy is an important pathological feature of several ocular diseases. Pharmacological manipulation of autophagy may provide an alternative therapeutic target for some ocular diseases. In this manuscript, we reviewed the relevant progress about the role of autophagy in the pathogenesis of ocular diseases.

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Abstract: Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms Recent reviews have described the range of assays that have been used for this purpose(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi) Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response

Global Epigenomic Reconfiguration During Mammalian Brain Development

Abstract: DNA methylation is implicated in mammalian brain development and plasticity underlying learning and memory. We report the genome-wide composition, patterning, cell specificity, and dynamics of DNA methylation at single-base resolution in human and mouse frontal cortex throughout their lifespan. Widespread methylome reconfiguration occurs during fetal to young adult development, coincident with synaptogenesis. During this period, highly conserved non-CG methylation (mCH) accumulates in neurons, but not glia, to become the dominant form of methylation in the human neuronal genome. Moreover, we found an mCH signature that identifies genes escaping X-chromosome inactivation. Last, whole-genome single-base resolution 5-hydroxymethylcytosine (hmC) maps revealed that hmC marks fetal brain cell genomes at putative regulatory regions that are CG-demethylated and activated in the adult brain and that CG demethylation at these hmC-poised loci depends on Tet2 activity.