Background

A stepwise epigenetic process controls immunoglobulin allelic exclusion. The functional Igμ

is expressed on the active allele, and the other allele is repressive.

Proto-oncogenes and IGH translocation is a common mechanism that drives B-ALL.

Rationale

IGH-DUX4: allelic specificity

DNA fragment assembly at

IGH-DUX4 translocation

region using Chromium

WGS and known sequence

near breakpoints.

Mapping Iso-Seq full tran-

script CCS reads (144,756

reads, median

length 4,350 bp).

Functional IGH and DUX4 related transcripts are assigned to different alleles accroding to 2

extronic heterozygous SNPs.

µ VH

DH

JH

Eµ

δ

rs1059713

A/T

rs1136534

A/G

rs3751511

A/G

IGH-DUX4 rearranged alleleIGHM DUX4 DUX4

DUX4

Enhancer

DUX4-IGH fusion transcript

IgH-DUX4 fusion transcript

antisense DUX4 transcript

non-functional IGH transcript

IGHJ4

T G G

Wild type alleleIGHM IGHJ6Enhancer IGHV1-69 Promoter

functional Igμ

A A A

The allele harbored IGH-DUX4

translocation is more repressive than

the wild type allele accroding to 3

heterozygous SNPs near IGHM, the

constant region of IGH without low

mappability issue.

Evidence from 3D genome

DUX4 regionEμ IGHV1-69IGHA1

IGHM

IGHEIGHJ4

IGHD4-17

IGHV3-21IGHV1-8

Hi-C

WGS

RNAseq

ATAC-seq

Capture-C

chr14chr10

Heterozygous SNPs in the IGH

promoter region were uniformly

mono-allelic in RNA-seq. In

Hi-C, H3K27ac HiChIP, and

Capture-C data, Eμ-IGH promoter contact reads were

uniformly mono-allelic from the

wild type allele.

Oncogenic fusion of IGH-DUX4 has recently been reported as a hallmark that defines a B-ALL

subtype present in up to 7% of adolescents and young adults B-ALL. The translocation of DUX4

into IGH results in aberrant activation of DUX4 by hijacking the intronic IGH enhancer (Eμ).

How IGH-DUX4 translocation interplays with IGH allelic exclusion was never been explored. We

investigated this in Nalm6 B-ALL cell line, using long-read (PacBio Iso-Seq method and 10X

Chromium WGS), short-read (Illumina total stranded RNA and WGS), epigenome (H3K27ac

ChIP-seq, ATAC-seq) and 3-D genome (Hi-C, H3K27ac HiChIP, Capture-C).

In most B-ALL patients with IGH-DUX4 or IGH-CRLF2 translocation, DUX4/CRLF2

expression is much lower than Igμ.

IGH@ patients

●

●

●●● ●● ●● ●

● ●●●●● ●

●●●

● ●●●

●10

100

500

2000

1000

10 100

FP

KM

of

Igμ

FPKM of CRLF2

●●

● ●

●

●●

●●●

●●●●

●●●●

●

●

●●

●

●

●

●

●

●

●

●●●●

●

●●

●1

10

100

500

1000

2000

1 10 100 500

FPKM of DUX4

FP

KM

of Igμ

●

●

●●

Cleavage

IGH−DUX4 BALL

Nalm6

In 28 out of 32 patients, Igμ to DUX4

FPKM fold>2, median 6.21

In 17 out of 24 patients, Igμ to CRLF2

FPKM fold>2, median 6.54

Conclusion

1 In Nalm6, IGH-DUX4 fusion occurred on the repressive allele that did not express

functional Igμ, and the fusion DUX4 exhibited lower expression compared to Igμ.

2 Epigenetic regulation at IGH locus is a plausible explanation as we observed weaker in-

teraction of Eμ-DUX4 than that of Eμ-IGH promoter.

3 It might be the same in B-ALL patients with IGH-DUX4 or IGH-CRLF2 translocation.

Question: Why does DUX4 translocate to the repressive IGH allele?

Highly expressed DUX4

is toxic to pre-B cell and

leads to cell death.

“High levels DUX4 induced

rapid cell death in 3T3 fibroblasts.”

The allele with DUX4

translocation will loss the

ability of successful

VDJ recombination

and expressing functional Igμ.

Model A:

Model B:

pro-B cell

pre-B cell

Highly expresed

DUX4

Igμ

DUX4 DUX4Igμ

pro-B cell

pre-B cell

Igμ

DUX4 DUX4VDJ recombionation

is failed, no functional Igμ

Cell death

500

Bosnakovski D et al. EMBO J. 2008

Bergman Y, Cedar H. Nat Rev Immunol. 2004

Hi-C and H3K27ac

HiChIP data

confirmed Eμ-IGH promoter interaction

and Eμ-DUX4 interaction.

Capture-C was

designed to identify

possible interactions

with Eμ. We estimated the

contact intensity of

Eμ-DUX4 was ~14-fold lower than

that of Eμ-IGH promoter. It may

explain the lower

expression of DUX4

compared to Igμ.

Ashby M et al. AACR Annual Meeting. 2017

ATAC-seq

Capture-C

Hi-C

Yasuda et al. Nat Genet. 2016

H3K27ac HiChIP

chr14chr10

Eμ IGHV1-69DUX4 region IGHA1IGHM

IGHEIGHJ4

IGHD4-17

IGHV3-21

IGHV1-8

Allelic specificity of immunoglobulin heavy chain (IGH@) translocation in B-cell acute lymphoblastic leukemia (B-ALL)

unveiled by long-read sequencingLiqing Tian1, Ying Shao1, Beisi Xu1, John Easton1, Xiaotu Ma1, Yongjin Li1, Scott Newman1, Xin Zhou1, Meredith Ashby2, Ting Hon2, Tyson Clark2, Elizabeth Tseng2 and Jinghui Zhang1

1 Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, Tennessee. 2 Pacific Biosciences, Menlo Park, California.

antisense DUX4

IGHM IGHJ6 D4Z4 repeats

IGHJ4

From assembled contig From hg38D4Z4 repeats

IGHD2-15

functional Igμ

IGH-DUX4 fusion

Iso-seq full

transcript CCS

Assembly

non-functional IGH

IGH-DUX4 rearranged allele

Wild type allele

Re

ad

s C

ove

rag

e

WG

S A

TAC−seq

RNAseq

rs1059713 rs1136534 rs3751511

0

10

20

30

40

0

10

20

0

5k

10k

15k

20k

0

10

20

30

40 H3

K2

7a

c

***

***

*

*

**

*

Full-length transcript sequencing

by Iso-Seq

IGH-DUX4 rearranged allele

Wild type allele

Eμ-IGH promoter interaction

Eμ-DUX4 interaction

One-tailed binomial test

(expected prob 0.5)

p<0.05

p<0.01

p<0.001

*

**

***

One-tailed binomial test

(expected prob 0.67)

p<0.01

p<0.001**

***

Eμ-DUX4 interaction Eμ-IGH promoter interaction

WG

S RNAseq

Hi−C H

iChIP

Capture−C

0

50

100

150

0 2.5k 5.0k 7.5k

01234

012345

0

200

400

600

Reads C

overa

ge

***

***

******

***

0

20

40

60 H3K

27ac

**

**

rs1141720 rs4448834 rs11623124

#1485