Genet 302 lec 49 & 50
What are the 2 main functions of a centromere?
To hold sister chromatids together with cohesin and to provide the site where kinetochores attach microtubules for chromosome segregation
How many microtubules are sufficient for a yeast chromosome centromere?
1
How many microtubules typically attach to a human chromosome centromere in this lecture?
About 10 to 40
What is a point centromere?
A centromere defined by a specific DNA sequence
What is a regional centromere?
A centromere spread over a larger chromosomal region rather than one short specific sequence
What kinds of organisms usually have regional centromeres?
Most multicellular eukaryotes
Are regional centromeres surrounded by repeat DNA?
Yes
Do the repeat sequences around Drosophila centromeres stay the same on every chromosome?
No, they vary between chromosomes
Do regional centromeres have one specific defining DNA sequence according to the mini-chromosome experiment?
No
What sequences were found in the sequenced Drosophila mini-centromere?
Satellite repeats such as (AATAT)n and (TTCTC)n plus clusters of transposable elements
According to the lecture, is repeat or satellite DNA enough to support centromere activity?
Yes, it is sufficient
According to the lecture, is repeat or satellite DNA also required for centromere activity?
Yes, it is necessary
What is the final testable statement about repeat DNA and centromeres?
Repeat or satellite DNA is both necessary and sufficient to make a centromere
What is CenH3?
A centromere-specific histone H3 variant
Where is regular H3 mainly found?
Euchromatin and most heterochromatin
Where is CenH3 found?
Centromeric heterochromatin
How was CenH3 experimentally targeted to a new chromosomal site in Drosophila cells?
A LacO DNA site was inserted and a LacR-CenH3 fusion protein was expressed to bind there
What happened when CenH3 was targeted to the LacO site?
Regular CenH3 was recruited there
What happened after regular CenH3 was recruited to the new site?
Kinetochore proteins were recruited there too
What conclusion came from the LacR-CenH3 targeting experiment?
CenH3 is necessary and sufficient to make a centromere
What are the three epigenetic mark types compared in lecture 50?
DNA methylation, histone modifications, and variant histones
How is DNA methylation maintained?
Maintenance methyltransferases act after DNA replication to copy methylation onto the new strand
How are histone modification marks maintained?
Reader-writer complexes act after DNA replication to copy the mark onto nearby new histones
How is the CenH3 variant-histone mark maintained?
A CenH3 assembly factor adds CenH3
When does the CenH3 assembly factor act?
Before DNA replication
Does centromeric DNA become only partially marked during mitosis in this model?
Yes
What are the three chromatin types you should compare in the lecture 50 diagram?
Centromeric heterochromatin, regular heterochromatin, and euchromatin
What is shared by centromeric heterochromatin and regular heterochromatin?
Both are compact chromatin states
What is unique to centromeric heterochromatin compared with regular heterochromatin?
It contains CenH3 and forms the kinetochore-bearing centromere
What is unique to regular heterochromatin compared with centromeric heterochromatin?
It lacks CenH3 and mainly functions in silencing/compaction rather than kinetochore formation
How does euchromatin differ from both types of heterochromatin?
It is less compact and more transcriptionally active
Why are regional centromeres flanked by extra repeat DNA according to one model?
To protect nearby genes from spread or shift of CenH3
Why are regional centromeres flanked by extra repeat DNA according to the Hendzel lab model?
Because repeat DNA strengthens the chromosome where microtubules apply force
What heterochromatin enzyme was tested for a role in mitosis around centromeres?
SU(VAR)3-9
What happened to mitosis when SU(VAR)3-9 was inhibited?
Mitosis failed, in specific metaphase- chromosmes can get pulled to the middle of the cell
Was CenH3 lost when SU(VAR)3-9 was inhibited?
No, CenH3 was unaffected- can still go to the centromere
Were microtubules affected when SU(VAR)3-9 was inhibited?
Yes, they try to connect but chromsomes cant withstand pulling force
What does the SU(VAR)3-9 inhibitor experiment suggest about centromeric heterochromatin?
It is important for proper microtubule function and chromosome segregation even though CenH3 can still be present
What simple comparison should your lecture 50 diagram show?
Euchromatin with regular H3 and active genes, regular heterochromatin with regular H3 and compaction/silencing, and centromeric heterochromatin with CenH3 plus kinetochore function