REGULOME – Lecture 4
Regulatory layers II
Part 4: transcription factors, methods to study chromatin
COO: ChIP-seq data analysis II
Transcription activation & transcription factors
RNA polymerase
Does transcription (DNA --> (non)coding RNA)
TSS = gene transcription start site, not a random place for start transcription
RNA polymerase has 10-12 subunits
Three types:
I --> ribosomal RNAs
II --> pre-mRNAs and most microRNAs
III --> tRNAs, rRNA, ssRNAs
o I and III have to be expressed in large amounts, in every cell, not so tightly regulated
o II has to be well regulated (mRNA determines cell type specificity)
More than half of the genome is actively transcribed --> genome very active (but in every cell
different regions are transcribed) (ChIP-seq of interaction between RNA polymerase and DNA)
RNA polymerase
General purpose transcriptional machinery
Cannot bind TSS by itself, requires interactions with other protein (mostly TFs)
Has to be recruited to the site by context specific proteins
Transcription Factors
Bind specific DNA sequences
10% of all genes are TFs
Dont bind randomly
Bind open DNA
Transcription Factor Binding Sites
Short stretches of DNA (4-20 bp)
Millions in the human genome
TFBS position weight matrix
Sequences not exactly defined, bit of flexibility on what the TF can recognize --> more
variability
Promoters
Regions of DNA (100-1000 bp)
, Immediately upstream of the TSS
Binding sites for general TFs, sometimes specific sites
As many promoters as TSS's, many genes have more than one TSS
Enhancers
Region of 100-1000 bp
Up to 1Mb or more upstream or downstream from the TSS
Binding sites for multiple TFs
Enhancer bound by TF --> turns on / accelerate transcription
Enhancer far away from promoter in sequence can get very close to promoter in space (DNA looping)
Gene regulatory domain
Full repertoire of enhances that affect the expression of a gene
Don't have to be contiguous in genome sequence
Can also regulate more than one gene
Transcription factors bind moslty to active enhancers (places in the DNA that are already open) -->
region even more active
Thus, activation by the tf depends on how active the region was before binding
Enhancers are cell type specific
A: every row is promotor, column is cell type
A few promotors are very cell specific, another set of promotors are active in every cell type
(housekeeping processes)
B: every row is enhancer, column is cell type
Enhancers are cell type specific
Enhancer landscape --> cell type specificity
Transcription factors have large 'fan outs'
One tf can regulate other tfs and so on (not one-to-one interactions in higher eukaryotes)
Activation of a single tf can lead to a change of the whole cell type! (thousands of target
genes and other transcription factors that are activated)
How to study enhancers: reporter systems
Let the enhancer control the expression of the reporter gene
Regulatory layers II
Part 4: transcription factors, methods to study chromatin
COO: ChIP-seq data analysis II
Transcription activation & transcription factors
RNA polymerase
Does transcription (DNA --> (non)coding RNA)
TSS = gene transcription start site, not a random place for start transcription
RNA polymerase has 10-12 subunits
Three types:
I --> ribosomal RNAs
II --> pre-mRNAs and most microRNAs
III --> tRNAs, rRNA, ssRNAs
o I and III have to be expressed in large amounts, in every cell, not so tightly regulated
o II has to be well regulated (mRNA determines cell type specificity)
More than half of the genome is actively transcribed --> genome very active (but in every cell
different regions are transcribed) (ChIP-seq of interaction between RNA polymerase and DNA)
RNA polymerase
General purpose transcriptional machinery
Cannot bind TSS by itself, requires interactions with other protein (mostly TFs)
Has to be recruited to the site by context specific proteins
Transcription Factors
Bind specific DNA sequences
10% of all genes are TFs
Dont bind randomly
Bind open DNA
Transcription Factor Binding Sites
Short stretches of DNA (4-20 bp)
Millions in the human genome
TFBS position weight matrix
Sequences not exactly defined, bit of flexibility on what the TF can recognize --> more
variability
Promoters
Regions of DNA (100-1000 bp)
, Immediately upstream of the TSS
Binding sites for general TFs, sometimes specific sites
As many promoters as TSS's, many genes have more than one TSS
Enhancers
Region of 100-1000 bp
Up to 1Mb or more upstream or downstream from the TSS
Binding sites for multiple TFs
Enhancer bound by TF --> turns on / accelerate transcription
Enhancer far away from promoter in sequence can get very close to promoter in space (DNA looping)
Gene regulatory domain
Full repertoire of enhances that affect the expression of a gene
Don't have to be contiguous in genome sequence
Can also regulate more than one gene
Transcription factors bind moslty to active enhancers (places in the DNA that are already open) -->
region even more active
Thus, activation by the tf depends on how active the region was before binding
Enhancers are cell type specific
A: every row is promotor, column is cell type
A few promotors are very cell specific, another set of promotors are active in every cell type
(housekeeping processes)
B: every row is enhancer, column is cell type
Enhancers are cell type specific
Enhancer landscape --> cell type specificity
Transcription factors have large 'fan outs'
One tf can regulate other tfs and so on (not one-to-one interactions in higher eukaryotes)
Activation of a single tf can lead to a change of the whole cell type! (thousands of target
genes and other transcription factors that are activated)
How to study enhancers: reporter systems
Let the enhancer control the expression of the reporter gene
