Chromatin Structure and Epigenetics and RNA

CNBY PPTs 11 and 12

What are chromosomes made of?

Chromosomes are composed of chromatin, which consists of 60% DNA, 5% RNA, and 35% protein

What is chromatin?

Chromatin is the complex of DNA and proteins that forms chromosomes; it organizes and packs DNA into the nucleus

What is meant by “chromosome territories”?

Specific regions in the nucleus where individual chromosomes are localized

What is a nucleosome?

The fundamental unit of chromatin — 147 base pairs of DNA wrapped 1.7 times around a histone core

What makes up the histone core?

An octamer of histones: two copies each of H2A, H2B, H3, and H4

How is chromatin often described visually?

As “beads on a string” — the beads are nucleosomes and the string is linker DNA

What is the main function of histones?

They package DNA into structural units (nucleosomes) and regulate gene accessibility through modifications

What are post-translational modifications (PTMs)?

Chemical changes to proteins after translation that alter their structure or function

What are the four common histone PTMs?

Phosphorylation, acetylation, methylation, and ubiquitination

Which amino acids are phosphorylated?

Tyrosine (Y), Serine (S), and Threonine (T)

What effect can phosphorylation have?

It can activate or inhibit protein function

Give an example of histone phosphorylation.

Histone H2A phosphorylation in response to DNA damage produces γH2AX

What happens during histone acetylation?

An acetyl group is added, relaxing chromatin and promoting gene expression

Which enzymes regulate acetylation?

HATs (Histone Acetyltransferases), HDACs (Histone Deacetylases)

HDACs (Histone Deacetylases 1)

1 Remove acetyl groups

HATs (Histone Acetyltransferases)

Add acetyl groups

How does acetylation affect lysine (K)?

Neutralizes its positive charge, weakening DNA binding and opening chromatin for transcription

What happens when HDACs remove acetyl groups?

Chromatin compacts again, reducing transcription; HDAC activity is needed for cell cycle progression

What do HDAC inhibitors cause?

Cell-cycle arrest

What is histone methylation?

Addition of one, two, or three methyl groups to amino acids, usually leading to transcriptional repression

What is the general function of methylation vs. acetylation?

Methylation, Acetylation

Acetylation

Gene activation

Methylation

Gene silencing 

What enzymes regulate methylation?

HMT (Histone Methyltransferase), HDM (Histone Demethylase)

HDM (Histone Demethylase 1)

1 Removes methyl groups

HMT (Histone Methyltransferase)

Adds methyl groups

Match the enzyme with its function: HAT

Adds acetyl groups

Match the enzyme with its function: HDAC

Removes acetyl groups

Match the enzyme with its function: HMT 

Adds methyl group

Match the enzyme with its function: HDM

Removes methyl groups

Match the enzyme with its function: DNMT

Adds methyl groups directly to DNA

How can histone-related mutations contribute to cancer?

Mutations in histones or modifying enzymes can alter gene regulation and drive oncogenesis

What are “oncohistones”?

Histone proteins with mutations, especially in the tails, that promote cancer

What are some enzymes that can be mutated in cancer?

HATs, HDACs, and other transferases

What histone mutations are linked to pediatric brain tumors?

Mutations in histone H3 — K27M or G34V/R

How do these mutations differ by tumor type?

Cortex/cerebrum (glioma) vs. midline/brainstem (DIPG) show different histone H3 mutations

What does the SWI/SNF complex do?

Controls chromatin remodeling, repositioning nucleosomes to regulate gene expression

How is SWI/SNF linked to cancer?

Mutations in its subunits are associated with various cancers

What is PBRM1?

A gene encoding the BAF180 protein, part of the SWI/SNF complex; acts as a tumor suppressor

What happens when PBRM1 is mutated?

It can contribute to tumorigenesis; it is one of the top mutated genes in some cancers

What does epigenetics mean?

It refers to changes to DNA that do not alter the nucleotide sequence but affect gene expression

What is the Greek meaning of “epi”?

“Around,” “over,” or “on top of” — referring to modifications on top of DNA

What is the most common epigenetic modification?

DNA methylation

How does DNA methylation affect gene expression?

It typically suppresses transcription and silences genes, especially when it occurs at promoter regions

Why is methylation important in normal cells?

It helps regulate differentiation by turning off genes no longer needed as cells specialize

Which nucleotides are naturally methylated in DNA?

Adenine and cytosine (cytosine is the most common)

Is epigenetic methylation the same as chemotherapy-induced methylation?

No. Chemotherapy-induced methylation (e.g., O-6 or N-7 methylguanine) damages DNA and must be repaired

Where does DNA methylation usually occur?

At CpG dinucleotides — cytosine followed by guanine linked by a phosphate bond

What percentage of CpG dinucleotides are methylated in somatic cells?

About 75%

What happens when promoter regions are methylated?

The associated genes are silenced and not expressed

What defines a CpG island?

• Length >200 base pairs

• G+C content >50%

  • Often found in gene promoter regions

What proportion of human genes have CpG islands in their promoter regions?

About 60–70%

What percentage of CpG islands are methylated in somatic tissues?

Only about 10%

What is global hypermethylation?

A widespread increase in DNA methylation seen in many cancers

What does CIMP stand for?

CpG Island Methylator Phenotype

What characterizes CIMP-positive tumors?

High levels of CpG island methylation — distinct from non-methylated tumors

What is the relationship between CIMP and microsatellite instability (MSI)?

CIMP+ tumors, especially in colon cancer, are often associated with MSI due to mismatch repair defects

How does CIMP status affect glioma prognosis?

G-CIMP (glioma CIMP) tumors are heavily methylated and have a better prognosis

How does methylation of tumor suppressor genes lead to cancer?

It silences tumor suppressor expression without changing the DNA sequence, promoting oncogenesis

Why might DNA sequencing miss epigenetic silencing?

Because sequencing detects mutations or deletions, not methylation

What special technique is needed to study methylation?

DNA methylation-specific sequencing

What is the goal of epigenetic therapy in cancer?

To decrease methylation and reactivate silenced tumor suppressor genes

Name two DNMT inhibitors used clinically.

• 5-Azacytidine: Approved for myelodysplastic syndrome and AML.

• Decitabine (5-aza-2’-deoxycytidine): Approved for myelodysplastic syndrome

What happens when the MGMT gene is methylated in glioblastoma?

MGMT is silenced, reducing repair of alkylation damage — making tumors more sensitive to chemotherapy

What happens when MLH1 is methylated in colon or endometrial cancer?

MLH1 is silenced (not mutated), leading to mismatch repair deficiency and increased cancer risk

What percentage of the human genome is coding DNA?

Only about 2%.

What does coding DNA do?

It is transcribed into mRNA and translated into protein

What is non-coding DNA?

DNA that does not encode proteins but can produce regulatory RNAs or serve as binding regions

What are introns and exons?

Exons are coding segments; introns are non-coding regions removed during RNA processing

What are enhancers?

DNA regions that bind activator proteins to boost transcription

What transcription factors can drive oncogenesis when overactive?

Myc, STAT3, NF-κB (p65), and AP-1 (Fos and Jun)

What transcription factor can suppress oncogenesis when lost?

p53

What are the main RNA types in translation?

mRNA (messenger), tRNA (transfer), rRNA (ribosomal)

mRNA (messenger)

Template for protein synthesis

tRNA (transfer)

Brings amino acids to the ribosome

rRNA (ribosomal)

Structural component of ribosomes

How can mRNA splicing affect cancer?

Abnormal splicing can alter tumor suppressor function

How are tRNA and rRNA linked to cancer?

Mutations or altered expression can increase translation efficiency in cancer cells

What are miRNAs? (microRNA)

Small (18–25 nt) noncoding RNAs that regulate mRNA translation by binding to target transcripts

How do miRNAs inhibit protein production?

By binding mRNA and promoting its degradation (deadenylation)

How do miRNAs differ from siRNAs?

• miRNAs are endogenous and only partially match targets.

  • siRNAs are exogenous, perfectly complementary, and used in labs or plants.

What enzymes process miRNA precursors?

Drosha and Dicer (endoribonucleases)

What is RISC?

RNA-induced silencing complex — where mature miRNAs guide mRNA degradation

What are OncomiRs?

miRNAs upregulated in cancers (e.g., miR-21, miR-155, miR-17-92)

What are tumor-suppressor miRNAs?

miRNAs downregulated in cancers (e.g., let-7, miR-34)

What is an AntagomiR (or antimiR)?

A synthetic RNA complementary to an oncomiR — used therapeutically to inhibit its function

What was MRX34?

An antagomiR targeting miR-34 tested in trials; halted due to adverse effects

What are lncRNAs?

Long noncoding RNAs (>200 nt) that regulate gene expression without encoding proteins

Where are lncRNAs found?

In both the nucleus and cytoplasm

Why are lncRNAs considered “genes”?

Because they have distinct, functional roles in cellular processes

Name four well-studied lncRNAs

HOTAIR, MEG3, MALAT1, NEAT1

HOTAIR

Overexpressed in breast and other cancers

MEG3

Decreased in colorectal and ovarian cancers

MALAT1

Metastasis-associated lung adenocarcinoma transcript

NEAT1

Nuclear paraspeckle assembly transcript

What is known about HOTAIR?

It’s 2158 nucleotides long and promotes breast cancer progression

What is known about MEG3?

It’s a tumor suppressor lncRNA; decreased expression correlates with advanced cancer

Are lncRNA-based therapies available?

Not yet — still in early development stages