2 LC Concepts of Molecular Biology 05.10.2022
Transcription and Regulation:
Epigenetics, histone modifications
Short recap:
• Pol II activity is highly regulated during initiation and transition into elongation
• TF are the main driver of gene expression
• Post-translational modifications on the Pol II CTD are important for regulating its function
One of the many obstacles for POL II: the nucleosome and its modifications
ChIP-Seq
Within the nucleus there are proteins that are binding to the DNA; they
can be well connected to it using formaldehyde and crosslinking them.
These crosslinks are stable during the next steps, in which DNS is taken
out and shred putting a lot of pression on it (ex. through sonication).
DNA is cut into smaller pieces about 200-500 bp basepairs.
These pieces of dna are connected to the proteins. using antibodies
specific against them and then antibodies themselves can be regonized
using protein G or protein A, that are binding to the heaving chain of
the antibody. DNA is then separated and sequenced using most of the
time next generation sequence technologies; reads are align to the
reference genome and it can be identified where the the proteins were
bound.
EPIGENETICS
epigenetics is the study of heritable changes in gene expression (active
versus inactive genes) that do nor involve changes to the underlying DNA
sequence. (most used, and but its better to talk about epigenetics heritability)
There are different definitions:
- Gene regulation
- Study of mechanisms that switch genes on or off
- Study of changes in organism caused by modification of gene expression rather alteration of the genetic code itself *
- Epigenetic literally means: in addition to chances in genetic sequence *
She defines it as a middle of these last two.
There is not a real clear definition.
One mark everyone can agree on as epigenetic mark is DNA methylation.
most higher eukayots in the context of CG, cytosine is methylated at the 5’C
atom. It chances the dna somehow permanently and it is a stable covalent
bound.
Enzymes involved:
De novo methylation: DNMT3a/b/l
Maintenance methylation: DNMT1 those marks after replication of dna, that have been on one strand are then trasfered to
the other strand.
Demethylation: TET1/2/3 removing methylation and going back to cytosine
IMPRINTING
, 2 LC Concepts of Molecular Biology 05.10.2022
It is when there are two different alleles differentially marked, in either the maternal or the paternal specific way. This is
happening when for example:
In this case the locus control region IC1, a strong enhancer that can be found next to the h19 gene when is methylated only on
the paternal allele. (lollipop methylated is filled in black).
Within the telomeric domain, enhancers (green ovals) direct transcription of the long non-coding RNA (lncRNA) H19 and
the intragenic microRNA (miRNA) miR-675 on the maternal chromosome (MAT; red line) and that of the growth factor
gene IGF2 and the intragenic miR-483 on the paternal chromosome (PAT; blue line). The imprinting centre of the
telomeric domain (IC1; inset), also known as the H19–IGF2 intergenic differentially methylated region (DMR), contains
tandem repeats (light red and light blue rectangles) and is bound by the transcription factors (TFs) CTCF, POU5F1 and
SOX2, which maintain the unmethylated status of the maternal allele, whereas ZFP57 maintains the methylated status
of the paternal allele. IC1 and IC2 are also characterized by different chromatin configurations on parental
chromosomes, with repressive histone marks, such as histone 3 lysine 9 dimethylation (H3K9me2), H3K9me3 and
H4K20me3 on the methylated allele, and permissive histone marks, such as H3K4me2 and H3K4me3, on the
unmethylated allele. Secondary DMRs (H19 promoter (prom), IGF2 DMR0 and IGF2 DMR2) are paternally methylated.
The imprinting centre of the centromeric domain (IC2), also known as KCNQ1OT1:transcription start site (TSS) DMR, is
maternally methylated and directs maternal-specific expression of KCNQ1 and the cell cycle regulator CDKN1C. On the
paternal allele, a lncRNA intragenic to KCNQ1 (KCNQ1OT1) is transcribed (wavy blue lines), suppressing in cis the
expression of coding genes in the region. IC2 methylation and silencing of the KCNQ1OT1 promoter are maintained
through interaction with ZFP57 on the maternal chromosome, while as yet uncharacterized TFs
sustain KCNQ1OT1 transcription on the paternal allele. Active alleles are represented with red (maternal) and blue
(paternal) oblong rectangles, and inactive alleles are represented with grey oblong rectangles.
During the germ cell development these methylations are put in place and in that specific case CTCF
cannot bind anymore. CTCF is a transcription factor involved in the 3D genome organization.
IGF2 in this case is the only one expressed from the paternal allele, not from the maternal because this
enhancer since its unmethylated can only activate the H19 gene.
So we have a difference in the maternal and the paternal genome, where we have really expression of
either on gene from mom or dad. This leads to changes in the 3d genome architecture, but it has
massive defects from the downstream expression pattern. ?
DNA methylation is very widely most of the CpGs in our genome are methylated, but when there are a
lot of CpGs for whatever reason they are not methylated. We don’t know why, probably because of
transcription factors that are keeping them from being methylated.
NUCLEOSOMES
Each DNA, 146 bp most of the times, are rapped around a set of proteins 8 core histones forming a
very stereotypical histone-octomia.
Transcription and Regulation:
Epigenetics, histone modifications
Short recap:
• Pol II activity is highly regulated during initiation and transition into elongation
• TF are the main driver of gene expression
• Post-translational modifications on the Pol II CTD are important for regulating its function
One of the many obstacles for POL II: the nucleosome and its modifications
ChIP-Seq
Within the nucleus there are proteins that are binding to the DNA; they
can be well connected to it using formaldehyde and crosslinking them.
These crosslinks are stable during the next steps, in which DNS is taken
out and shred putting a lot of pression on it (ex. through sonication).
DNA is cut into smaller pieces about 200-500 bp basepairs.
These pieces of dna are connected to the proteins. using antibodies
specific against them and then antibodies themselves can be regonized
using protein G or protein A, that are binding to the heaving chain of
the antibody. DNA is then separated and sequenced using most of the
time next generation sequence technologies; reads are align to the
reference genome and it can be identified where the the proteins were
bound.
EPIGENETICS
epigenetics is the study of heritable changes in gene expression (active
versus inactive genes) that do nor involve changes to the underlying DNA
sequence. (most used, and but its better to talk about epigenetics heritability)
There are different definitions:
- Gene regulation
- Study of mechanisms that switch genes on or off
- Study of changes in organism caused by modification of gene expression rather alteration of the genetic code itself *
- Epigenetic literally means: in addition to chances in genetic sequence *
She defines it as a middle of these last two.
There is not a real clear definition.
One mark everyone can agree on as epigenetic mark is DNA methylation.
most higher eukayots in the context of CG, cytosine is methylated at the 5’C
atom. It chances the dna somehow permanently and it is a stable covalent
bound.
Enzymes involved:
De novo methylation: DNMT3a/b/l
Maintenance methylation: DNMT1 those marks after replication of dna, that have been on one strand are then trasfered to
the other strand.
Demethylation: TET1/2/3 removing methylation and going back to cytosine
IMPRINTING
, 2 LC Concepts of Molecular Biology 05.10.2022
It is when there are two different alleles differentially marked, in either the maternal or the paternal specific way. This is
happening when for example:
In this case the locus control region IC1, a strong enhancer that can be found next to the h19 gene when is methylated only on
the paternal allele. (lollipop methylated is filled in black).
Within the telomeric domain, enhancers (green ovals) direct transcription of the long non-coding RNA (lncRNA) H19 and
the intragenic microRNA (miRNA) miR-675 on the maternal chromosome (MAT; red line) and that of the growth factor
gene IGF2 and the intragenic miR-483 on the paternal chromosome (PAT; blue line). The imprinting centre of the
telomeric domain (IC1; inset), also known as the H19–IGF2 intergenic differentially methylated region (DMR), contains
tandem repeats (light red and light blue rectangles) and is bound by the transcription factors (TFs) CTCF, POU5F1 and
SOX2, which maintain the unmethylated status of the maternal allele, whereas ZFP57 maintains the methylated status
of the paternal allele. IC1 and IC2 are also characterized by different chromatin configurations on parental
chromosomes, with repressive histone marks, such as histone 3 lysine 9 dimethylation (H3K9me2), H3K9me3 and
H4K20me3 on the methylated allele, and permissive histone marks, such as H3K4me2 and H3K4me3, on the
unmethylated allele. Secondary DMRs (H19 promoter (prom), IGF2 DMR0 and IGF2 DMR2) are paternally methylated.
The imprinting centre of the centromeric domain (IC2), also known as KCNQ1OT1:transcription start site (TSS) DMR, is
maternally methylated and directs maternal-specific expression of KCNQ1 and the cell cycle regulator CDKN1C. On the
paternal allele, a lncRNA intragenic to KCNQ1 (KCNQ1OT1) is transcribed (wavy blue lines), suppressing in cis the
expression of coding genes in the region. IC2 methylation and silencing of the KCNQ1OT1 promoter are maintained
through interaction with ZFP57 on the maternal chromosome, while as yet uncharacterized TFs
sustain KCNQ1OT1 transcription on the paternal allele. Active alleles are represented with red (maternal) and blue
(paternal) oblong rectangles, and inactive alleles are represented with grey oblong rectangles.
During the germ cell development these methylations are put in place and in that specific case CTCF
cannot bind anymore. CTCF is a transcription factor involved in the 3D genome organization.
IGF2 in this case is the only one expressed from the paternal allele, not from the maternal because this
enhancer since its unmethylated can only activate the H19 gene.
So we have a difference in the maternal and the paternal genome, where we have really expression of
either on gene from mom or dad. This leads to changes in the 3d genome architecture, but it has
massive defects from the downstream expression pattern. ?
DNA methylation is very widely most of the CpGs in our genome are methylated, but when there are a
lot of CpGs for whatever reason they are not methylated. We don’t know why, probably because of
transcription factors that are keeping them from being methylated.
NUCLEOSOMES
Each DNA, 146 bp most of the times, are rapped around a set of proteins 8 core histones forming a
very stereotypical histone-octomia.
