The regulatory role and environmental sensitivity of DNA methylation in neurodevelopment

Indiana University-Purdue University Indianapolis (IUPUI)The emerging field of epigenetics is expanding our understanding of how biological diversity is generated in the face of genetic limitations. One epigenetic mechanism in particular, DNA methylation, has demonstrated a dynamic range during neural development. Here, we provide evidence that DNA methylation occurs as a cell unique program aiding in the regulation of neurodevelopmental gene expression. DNA methylation has demonstrated sensitivity to external inputs ranging from stress to chemical exposure and dietary factors. To explore DNA methylation as a means of communicating early-life stress to the brain, we utilized a mouse model of fetal alcohol spectrum disorders (FASD). FASD presents a range of neurodevelopmental deficits and is a leading cause of neurodevelopmental disabilities in the United States. Predicated on the knowledge of alcohol's teratogenic role in brain development, we describe that the normal pattern of cortical DNA methylation and epigenetic correlates is similarly impacted by prenatal alcohol exposure. Due to the biochemical interaction of alcohol metabolism and the pathways regulating DNA methylation synthesis, we further investigated whether dietary manipulation could normalize the cortical DNA methylation program and aid in the protection of FASD characteristics. We found that the alcohol sensitive DNA methylation landscape is dually capable of registering dietary intervention, demonstrating normalization of disease-related patterns in the cortex and improved neurodevelopmental gene expression and morphology. Finally, we investigated the DNA methylation landscape in a crucial corticodevelopmental gene to more accurately define the breadth and scope of the environmental impacts at the nucleotide level. We found that alcohol and dietary supplementation are selective for regions associated with transcriptional control. Collectively, the evidence supports that DNA methylation plays a regulatory role in development and that its sensitivity to external inputs is dynamic and detectable at the smallest genomic level. Importantly, DNA methylation landscapes are adaptable and thus bear diagnostic and therapeutic potential

DNA Methylation program in normal and alcohol-induced thinning cortex

While cerebral underdevelopment is a hallmark of fetal alcohol spectrum disorders (FASD), the mechanism(s) guiding the broad cortical neurodevelopmental deficits are not clear. DNA methylation is known to regulate early development and tissue specification through gene regulation. Here, we examined DNA methylation in the onset of alcohol-induced cortical thinning in a mouse model of FASD. C57BL/6 (B6) mice were administered a 4% alcohol (v/v) liquid diet from embryonic (E) days 7–16, and their embryos were harvested at E17, along with isocaloric liquid diet and lab chow controls. Cortical neuroanatomy, neural phenotypes, and epigenetic markers of methylation were assessed using immunohistochemistry, Western blot, and methyl-DNA assays. We report that cortical thickness, neuroepithelial proliferation, and neuronal migration and maturity were found to be deterred by alcohol at E17. Simultaneously, DNA methylation, including 5-methylcytosine (5mC) and 5-hydroxcylmethylcytosine (5hmC), which progresses as an intrinsic program guiding normal embryonic cortical development, was severely affected by in utero alcohol exposure. The intricate relationship between cortical thinning and this DNA methylation program disruption is detailed and illustrated. DNA methylation, dynamic across the multiple cortical layers during the late embryonic stage, is highly disrupted by fetal alcohol exposure; this disruption occurs in tandem with characteristic developmental abnormalities, ranging from structural to molecular. Finally, our findings point to a significant question for future exploration: whether epigenetics guides neurodevelopment or whether developmental conditions dictate epigenetic dynamics in the context of alcohol-induced cortical teratogenesis

Women in the area of health and science on the border of Mexico between Tamaulipas and Texas

Background: The border region between Mexico and Texas configures the space of binational, industrial, commercial and mercantile development, with great business openness on both sides of the border, where the cultural environment is marked by the altered way in which people develop on the border, where the man mostly exercises professional profiles related in the manufacturing maquiladoras area, industrial park, etc. while women are configured as professionals mostly in the area of health services, education and others, but within this space women make their way to science within the development of scientific research, without being identified for the sake of the border, which are usually culturally not associated with the imaginary of the border. Knowing the incursion into this geographical region on both sides of the border, can strengthen and promote the development of women in science and scientific developments, but on addressing the gender gaps in this sector that can also be addressed binationally. Case presentation: One of the main reasons why we want to participate, is due to the need to expose the professional practice of women in the area of health who live in the region of the border border formed between Reynosa, Tamaulipas and the Texas Valley towards Science. With the aim of transmitting the way in which the female gender stands out in the areas of scientific research within the national system of researchers and the gaps of opportunity for early training towards science in the border of Tamaulipas. We consider important the dissemination of information within the event, given that the research and development tasks in science in the border area is developed by women breaking professional stereotypes, but also promoting the path to training in science in early training. Conclusions: Women currently form part of 30% of the total number of researchers in the world, Mexico the participation of women in science is 37%, in the national system of researchers in Mexico there are 33, 166 women in the various areas of knowledge, distinguishes the percentage of women in activities dedicated to health in (medicine) , public health, ext.) In the mexican Republic and even more in the border territoriality in Tamaulipas, it will allow to know and distinguish the gender gaps for the strengthening of the border entity

Cell-Wide DNA De-Methylation and Re-Methylation of Purkinje Neurons in the Developing Cerebellum

Global DNA de-methylation is thought to occur only during pre-implantation and gametogenesis in mammals. Scalable, cell-wide de-methylation has not been demonstrated beyond totipotent stages. Here, we observed a large scale de-methylation and subsequent re-methylation (CDR) (including 5-methylcytosine (5mC) and 5-hydroxylmethylcytosine (5hmC)) in post-mitotic cerebellar Purkinje cells (PC) through the course of normal development. Through single cell immuno-identification and cell-specific quantitative methylation assays, we demonstrate that the CDR event is an intrinsically scheduled program, occurring in nearly every PC. Meanwhile, cerebellar granule cells and basket interneurons adopt their own DNA methylation program, independent of PCs. DNA de-methylation was further demonstrated at the gene level, on genes pertinent to PC development. The PC, being one of the largest neurons in the brain, may showcase an amplified epigenetic cycle which may mediate stage transformation including cell cycle arrest, vast axonal-dendritic growth, and synaptogenesis at the onset of neuronal specificity. This discovery is a key step toward better understanding the breadth and role of DNA methylation and de-methylation during neural ontology

Cell-Specific DNA Methylation profiles of the post-natal cerebellum.

<p>(<b>A</b>) 5mC-immunostaining (im) is intensively present in the nucleus (size >12μm) of postmitotic Purkinje Cells (PC) at P7 (red, crossed arrows) at PC layer (PCL). 5mC is also distinctively present only in the non-dividing granule cells of the inner portion of the External Granule Layer (EGLi, blue crossed arrows) but not outer portion of EGL (EGLo). (<b>B)</b> By P28, the waning of 5mC-im was evident in PCs (red, dashed circles; ~20μm diameter). Basket cells surrounding the PCs (purple dots, <8μm) were intensively immunostained by 5mC (as well as all other interneurons). Mature granule cells inhabiting the Inner Granule Layer (IGL) retained the acquired 5mc-im throughout the remainder of the time-course. <b>(C)</b> By P45 re-methylation of PCs occurred as the 5mC-im returned to some but not to all de-methylated PCs (red, crossed arrows denote re-methylated PCs). (<b>D</b>) At P7, 5hmC immunostaining (im) is intensively present in PCs (red, crossed arrows), though distributed distinctly from 5mC. Some granule cells of the inner EGL express 5hmC-im though not at the upper surface of the EGL, where granule cell progenitors reside. <b>(E)</b> At P28, a clear de-methylation of 5hmC occurs in the PCs (red, dashed circles) as occurs with 5mC. Granule cells which have migrated to the IGL continue to acquire 5hmC (blue, crossed arrows) as do the emerging basket interneurons surrounding the perimeter of PCs (purple dots). (<b>F</b>) At P45, re-methylation of 5hmC occurs in line with 5mC re-methylation at PCs (red, crossed arrows denote re-methylated PCs). Interneurons and granule cells appear to refrain from de-methylation throughout their developmental time-course. Scale bars: <b>A-F</b> = 20μm; Methyl Green Nissl counterstain. Dashed red circles depict the approximate boundaries of the PC cell body. Dashed black lines depict approximate boundaries of the PCL.</p

Postnatal loss of 5hmC is synchronized with loss of downstream 5fC and 5caC in PCs.

<p>5fC and 5caC are downstream metabolites of 5hmC (catalyzed by Tet enzymes) and prevail throughout cerebellar neurons including PCs (red, crossed arrow) and post-mitotic granule cells at P7 (<b>A,C</b>). As 5hmC is de-methylated at P21 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162063#pone.0162063.g001" target="_blank">Fig 1</a>), 5fC and 5caC are also greatly reduced (<b>B, D</b>, red arrows). On the other hand, as PCs undergo de-methylation of the 5fC and 5caC derivatives, surrounding basket cells acquire immunoreactivity. IGL: internal granule cell layer, PCL: Purkinje cell layer, ML: molecular layer, Nissl counterstaining: methyl green. Scale bar: <b>A-B</b> = 50μm, <b>C-D</b> = 20μm.</p

Quantitative detection of DNA methylation in the postnatal cerebellum.

<p>(<b>A</b>) <i>Diagram of Grid2 gene structure</i>. Transcription start site (TSS): black bent arrow; exons: black boxes. <i>Grid2</i> is transcribed from the Watson strand. A region in the intron 4 was analyzed. The lower half of the panel is a magnification of the target region. Three CpG dinucleotides and one <i>HhaI</i> cleavage site are located in the target region (Chr6:64,015,530–64,015,858). Displaying primer (blue, straight arrow), CpG dinucleotides (white “lollipops”), and restriction enzyme cleavage sites (red, dashed lines). (<b>B</b>) <i>Analysis of Grid2</i>: This figure represents DNA methylation changes between P7 and P29 following digestion with <i>HhaI</i>. The P7 cerebellum is highly methylated, while the P29 cerebellum shows about a 60% reduction in DNA methylation. P-value = 0.0037. <i>Gene expression of Grid2</i>: Quantitative RT-PCR of P7 and P29. <i>Grid2</i> is expressed 3.3 fold higher in P29 than in P7 cerebellum. P-value = 0.0006. (<b>C</b>) <i>Analysis of Syt2</i>. We amplified the following genomic region for <i>Syt2</i>: Chr1: 136603663+136603902. (<b>D</b>) This figure represents DNA methylation changes between P7 and P29 following digestion with <i>Hpa</i>II. The P7 cerebellum is almost completely methylated, while P29 cerebellum shows about a 60% reduction in DNA methylation. P-value = 0.045. From P7 to P29, <i>Syt2</i> mRNA expression is increased 4.6 –fold. (<b>E</b>) <i>Analysis of Cacna1g</i>. We amplified the following genomic region for <i>Cacn1g</i>: Chr11: 94336884–94337233. (<b>F</b>) This figure represents DNA methylation changes between P7 and P29 following digestion with <i>Hpa</i> II. The P7 cerebellum is ~ 72% methylated, while P29 cerebellum shows only 10% methylation. P-value = 0.0003. Gene body methylation analysis reveals a reciprocal relationship across ages, where P7 methylation begins around 10% and spikes to about 80% by P29. Overall gene expression was increased 2-fold across this time.</p

De-methylation and re-methylation are synchronized upon turnover of DNMT1 and Tet1 throughout PC maturation.

<p>The peak heterochromatic appearance of 5mC-im (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162063#pone.0162063.g001" target="_blank">Fig 1</a>) occurs at the same time as peak Dnmt1-im (<b>A</b>, red crossed arrows). Similarly, the peak euchromatic staining of 5hmC-im (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162063#pone.0162063.g001" target="_blank">Fig 1</a>) occurs at the same time as peak Tet1-im (<b>D</b>, red crossed-arrows) in PCs at P7. De-methylation follows progressively, as by P21 many PCs lacked DNMT1 (<b>B</b>, red arrows), and subsequently were devoid of Tet1 (<b>E</b>, red arrows). The methyl green counterstaining reveals 5hmC negative and Tet1-negative PC cell bodies (red arrows). Meanwhile, surrounding basket cells (and other interneurons) acquire Tet1 (<b>E</b>, purple dots). By P45, as re-methylation of 5mC is occurring, DNMT1-im is notably returned to the PC nuclei (<b>C</b>, red crossed-arrows). Similarly, Tet1 is observed parallel with the resumed observation of DNMT1-im expression (<b>F</b>, red crossed arrows) in PCs. PCL: Purkinje layer; EGL: external granule layer; IGL: internal granule layer. Scale bars: <b>A-C</b> = 20μm; <b>D-F</b> = 20μm.</p

Quantitative Detection of Cell-Specific, Developmental DNA Methylation in the Cerebellum.

<p>Purified gDNA obtained from laser micro-dissected Purkinje and Granule cells was quantitatively analyzed for 5-methylcytosine and 5-hydroxymethylcytosine content (%) via antibody-based colorimetric assay. (<b>A-B</b>) Purkinje cells undergo remarkable loss of both 5mC and 5hmC between the first and fourth post-natal weeks, coincident with the Purkinje cell morphological and transcriptional transformation. (<b>C-D</b>) Granule cells of the external granule surface, as they undergo radial migration into the internal granule layer and become post-mitotic, acquire 5mC as indicated by earlier immunohistochemical analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162063#pone.0162063.g001" target="_blank">Fig 1</a>). Further, granule cells of the internal granule layer (IGL) continue to acquire methylation between the first and fourth postnatal week as granule cells settle into their mature state. All values represented as mean ± SEM (<b>A</b>.) **P-value = 0.0078; (<b>B</b>.) ***P-value = 0.0001; N = 4 per age. (<b>C</b>.) *P-value = 0.0186; (<b>D</b>.)** P-value = 0.0036; N = 8 per age.</p

DNA Methylation Analysis by Restriction Enzyme Digestion Followed by qPCR.

<p>DNA Methylation Analysis by Restriction Enzyme Digestion Followed by qPCR.</p